Compositions and methods for regulating gene expression for targeted mutagenesis

ABSTRACT

Disclosed herein are methods for gene targeting in a plant cell, as well as recombinant nucleic acid molecules used in these methods. Further disclosed are recombinant nucleic acid molecules comprising a target gene operably linked to a regulatory region of the UBQ10 gene.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part Application of International Application No. PCT/IL2017/051020 filed Sep. 11, 2017 and published as WO 2018/047183 Mar. 15, 2018, which claims priority of Israel Patent Application No. 247752, filed Sep. 11, 2016. Each of the above listed documents is hereby incorporated by reference in its entirety.

FIELD OF DISCLOSURE

Disclosed herein are compositions and methods for regulating gene expression for gene targeting in eukaryotes, including plants and algae. Specifically, disclosed herein are recombinant nucleic acid molecules having the adjacent regions of POLYUBIQUITIN10 gene and uses thereof for enhancing the expression of a gene. Also disclosed here are methods for gene targeting in a plant cell, via homologous or non-homologous DNA repair and recombinant nucleic acid molecules used in these methods. The recombinant nucleic acid molecules comprise a viral replicon comprising a donor nucleic acid sequence and a nucleic acid comprising a nuclease system operably linked to a regulatory region of the UBQ10 gene.

BACKGROUND

The recently developed CRISPR-CAS system allows performing targeted gene modifications in most organisms. In plants, this technology can be used to knock-out specific genes to improve commercial traits. The limitation of this technology is its relatively low efficiency. To ensure highest efficiency, the CAS9 gene must be expressed at high levels in a stable manner. This is usually achieved by using the obvious cauliflower mosaic virus (CaMV) 35S promoter. This promoter is a viral promoter and is widely used in the field of plant sciences in both dicots and monocots. However, this promoter is often subjected to silencing by the plant machinery, which results in the low expression of the adjacent gene, and is even inactive in certain cell types, such as pollen. Surprisingly, this promoter is still the promoter of choice used during research in the plant science field.

Recently, the CRISPR-CAS system was developed and allows the mutagenesis of any given genes. To this end, one must express the CAS9 gene together with a guide RNA (gRNA) that can recognize the desired target. As a consequence, CAS9 cleaves the genomic DNA, and the DNA repair machinery restores the chromosome, typically inserting or deleting a few nucleotides. This causes a frame shift at the target site and prevents correct gene expression.

Expression of the CAS9 gene under a low-efficiency promoter results in low mutagenesis success. Consequently, in order to obtain the desired homozygous mutant, one has to first screen for the mutation, select the mutant plants, and further cross the mutant plants several times. Due to the long-life cycle of most plants species (several months at least), this procedure may take several months or years depending on the zygosity of the plant species of interest.

A recent work reported on gene targeting in tomato plants in the absence of mutant plants selection. The reported rates of targeted mutations were low, and only 1 plant had germinally transmitted mutations (Yu et al. 2017, Sci. Rep., 1-18). Other works in rice showed very low rates of germinally transmitted gene mutations using Cas9 and Cpf1.

Several approaches have been developed throughout the years to increase the frequency of homologous recombination between a genomic target and an extrachromosomal homologous donor. These include the expression of genes involved in homologous recombination (HR) that can increase rates of gene targeting (GT), the use of single stranded donor DNA, and the use of viral vectors albeit with limited success.

Inducing a DSB in a certain genomic region was shown to increase the rate of repair by HR with an Agrobacterium-delivered T-DNA vector. The increase was of 2-3 orders of magnitudes, but the GT rate remained very low, in the 10⁻²-10⁻³ range. This might be due to the competition with the more efficient non-homologous end-joining (NHEJ) repair pathway or due to low DNA DSB efficiency. Gene replacement can also be achieved via NHEJ through DSB-mediated excision of the targeted locus and insertion of the new donor DNA into the empty site. An improvement of the DSB-induced GT, called In planta GT approach, consists in the coordinated induction of a DSB in the genomic target and excision of the donor repair template from the plant DNA where it had been previously introduced.

Viral vectors are another approach that has been used for genetic manipulation of plants and for GT. Plant RNA viruses like the potato virus X (PVX) and the tobacco rattle virus (TRV) are commonly used for gene silencing and were also used for delivering ZFN nucleases or the CRISPR guide RNA (gRNA). Similarly, the Cabbage Leaf Curl virus was used for gRNA delivery in Nicotiana Benthamiana. These viruses spread efficiently in the plant yet the addition of an exogenous sequence to their genome is very limited due to size constraints of the viral particles, thus delivering the sequence of large proteins like Cas9 is not feasible.

Plant viruses from the geminivirus family have a DNA genome that replicates by a rolling circle replication (RCR) mechanism. Their genes can be transcribed within the plant nucleus, generating the proteins necessary for the RCR and for encapsidation to form virions. The ability of the virus to move from cell-to-cell (systemic infection) can be lost through insertion of DNA fragments whose length prevents encapsidation. For example, the maize streak virus (MSV) have shown to tolerate insertions in their genome and deliver DNA fragments to the plant, yet their systemic movement was damaged.

An engineered geminiviral replicon, which can undergo RCR but is not infective, was recently developed to deliver large sequences such as sequence specific nucleases (SSNs) and DNA repair templates, based on the bean yellow dwarf virus (BeYDV). This system was used in tomato to replace an endogenous promoter upstream of the ANTI gene with the cauliflower mosaic virus 35S promoter, which promotes constitutive anthocyanin synthesis serving as a marker for GT as early as in the stage of the calli formation. The BeYDV replicon was also used for generating targeted mutations and gene targeting in potato and cassava, and two different geminiviruses, the wheat dwarf virus (WDV) and the tomato leaf curl virus (ToLCV) were used for GT in hexaploid wheat, with WDV used for GT in rice.

Overall, the efficiency of GT achieved by the methods described above was relatively low or had to rely on markers, such as resistance (glyphosate), GFP, RFP, ANTI etc. This is not amenable to agricultural and research applications, where there is a need of a routine method with no addition of exogenous selectable or reporter markers. However, only a highly efficient GT would allow breeding transgenic plants without the need of selection markers.

Therefore, there is an unmet need for, and it would be highly advantageous to have means and methods for highly efficient GT in a plant.

SUMMARY OF THE DISCLOSURE

In one aspect, disclosed herein is a method for gene targeting in a plant cell, the method comprising: (a) introducing into said plant cell a first nucleic acid comprising a viral replicon comprising a donor nucleic acid sequence, said donor sequence targeted to a plant endogenous DNA sequence; and (b) introducing into said same plant cell a second nucleic acid comprising a nuclease system, wherein said nuclease system is targeted to said plant endogenous DNA sequence, and wherein at least one component of said nuclease system is expressed under a UBQ10 regulatory sequence; wherein homologous recombination occurs between the donor sequence and said plant endogenous DNA sequence.

In a related aspect, the viral replicon is selected from a group comprising: a geminiviral replicon, a bean yellow dwarf virus (BeYDV) replicon, a cabbage leaf curl virus (CalCuV) replicon, a tomato leaf curl virus (ToLCV) replicon, a wheat dwarf virus (WDV) replicon, or any combination thereof. In a related aspect, the donor sequence comprises a gene, a mutated gene, a part of a gene, a regulatory sequence, a mutated regulatory sequence, a sequence upstream of a gene, a sequence downstream of a gene, an exon sequence, an intron sequence, or any combination thereof.

In a related aspect, the nuclease system is selected from a group comprising: a nickase, a zinc finger nuclease (ZFN) system, a transcription activator-like effector nuclease (TALEN) system, a meganuclease, or a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) nuclease. In a related aspect, the CRISPR/Cas nuclease system comprises a CRISPR-associated endonuclease (Cas) and a gRNA molecule, wherein said gRNA molecule binds within said plant endogenous DNA sequence.

In a related aspect, the Cas enzyme is selected from the group comprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, C2c1, CasX, NgAgo, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4, or homologs thereof, or modified versions thereof. In a related aspect, the Cas is operably linked to said UBQ10 regulatory sequence.

In a related aspect, the single expression vector comprises said first nucleic acid and said second nucleic acid. In a related aspect, the UBQ10 regulatory sequence is selected from a group comprising: Solanum lycopersicum, Solanum tuberosum, or Arabidopsis thaliana Ubi10 regulatory sequence. In a related aspect, the Solanum lycopersicum UBQ10 regulatory sequence comprises Solanum lycopersicum UBQ10 promoter and terminator regions. In a related aspect, the gene targeting comprises gene editing, gene replacement, or a combination of both.

In one aspect, disclosed herein is a recombinant nucleic acid molecule comprising a first nucleotide sequence encoding a nuclease system, wherein said nuclease system is targeted to a plant endogenous DNA sequence, and wherein at least one component of said nuclease system is operably linked to a UBQ10 regulatory sequence.

In a related aspect, the recombinant nucleic acid molecule further comprises a second nucleotide sequence encoding a viral replicon comprising a donor nucleic acid sequence targeted to said plant endogenous DNA sequence.

In one aspect, disclosed herein is a method for producing a transgenic plant seed, the method comprising: (a) introducing into at least one cell of a plant a first nucleic acid comprising a viral replicon comprising a donor nucleic acid sequence, said donor sequence targeted to a plant endogenous DNA sequence; and (b) introducing into the cell of (a) a second nucleic acid comprising a nuclease system, wherein said nuclease system is targeted to said plant endogenous DNA sequence, and wherein at least one component of said nuclease system is expressed under a UBQ10 regulatory sequence: (c) generating a transgenic plant from said at least one cell; and (d) growing said transgenic plant to obtain a seed; wherein homologous recombination occurs between the donor sequence and said plant endogenous DNA sequence; thereby producing a transgenic seed of a plant, wherein any plant produced from said seed comprises said donor nucleic acid sequence.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1. RFP fluorescence observed in N. benthamiana leaves 7 days post inoculation, with Agrobacterium containing a plasmid that harbor the RFP gene under the control of various promoters and terminators. 35S: cauliflower mosaic virus (CaMV)—control; SICABI: CHLOROPHYLL A-B BINDING PROTEIN (Solyc02g071010)—SICABI promoter region (SEQ ID NO: 8)/SITHI4 terminator region (SEQ ID NO: 21); SCAB: CHLOROPHYLL A-B BINDING PROTEIN (Solyc03g005760)—SICAB promoter region (SEQ ID NO: 11)/SITHI4 terminator region (SEQ ID NO: 21); SIGAPDH: GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (Solyc04g009030)—SIGAPDH promoter region (SEQ ID NO: 14)/SITHI4 terminator region (SEQ ID NO: 21), SIMETALL: TYPE 2 METALLOTHIONEIN (Solyc09g010800)—SIMETALL promoter region (SEQ ID NO: 17)/SITHI4 terminator region (SEQ ID NO: 21); SlUBIQ10: POLYUBIQUITIN10 (Solyc07g064130)—SlUBIQ10 promoter region (SEQ ID NO: 23)/SlUBIQ10 terminator region (SEQ ID NO: 27): SIRBCS: RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL CHAIN (Solyc03g034220)—SIRBCS promoter region (SEQ ID NO: 31)/SITHI4 terminator region SEQ ID NO: 21).

FIG. 2. RFP fluorescence observed in N. benthamiana leaves 7 days post inoculation, with Agrobacterium containing a plasmid that harbor the RFP gene under the control of various promoters and terminators. SlUBIQ10: POLYUBIQUITIN10 of tomato (Solyc07g064130— SlUBIQ10 promoter region (SEQ ID NO: 23)/SlUBIQ10 terminator region (SEQ ID NO: 27); StUBIQ10: POLYUBIQUITIN10 of potato (Sotub07g026130—StUBIQ10 promoter region (SEQ ID NO: 34)/StUBIQ10 terminator region (SEQ ID NO: 38)): AtUBIQ10: POLYUBIQUITIN10 of Arabidopsis (AT4G05320—AtUBIQ10long promoter region (SEQ ID NO: 41)/AtUBIQ10long terminator region (SEQ ID NO: 45) and AtUBIQ10short promoter region (SEQ ID NO: 48)/AtUBIQ10short terminator region (SEQ ID NO: 45).

FIG. 3. Relative expression level of the VANILIN SYNTHASE gene of Capsicum annum (CaVAN) in transgenic tomatoes obtained in Example 3. (wt—wild type plant; Lines 1, 2, 3, and 4 tomatoes transformed using the CaVAN expression vector wherein CaVAN gene expression is under the control of the SlPOLYUBIQUITIN10 cassette (SlUBIQ10 promoter region (SEQ ID NO: 23)/SlUBIQ0 terminator region (SEQ ID NO: 27) CaVAN-ox—CaVAN-overexpression).

FIGS. 4A-4B. Relative expression level of the CAS9 gene in transgenic tomatoes obtained in Example 2, measured by (qRT-PCR). FIG. 4A shows CAS9 expression in M82 tomatoes. wt—wild-type plant. Lines 4, 5, 6, 7, 9, 11, 13, 14, and 15—M82 tomatoes transformed with the CAS9 expression vector wherein CAS9 gene expression is under the control of the SlPOLYUBIQUITIN10 cassette (SlUBIQ10 promoter region (SEQ ID NO: 23)/SlUBIQ10 terminator region (SEQ ID NO: 27)). FIG. 4B shows CAS9 expression in microtom tomatoes. wt—wild-type plant (CAS9 gene was expressed in a SlUBIQUITIN10 cassette). Lines 5, 6, 9, 12, 14, 17, and 19—microtom tomatoes transformed with the CAS9 expression vector wherein CAS9 gene expression is under the control of the SlPOLYUBIQUITIN10 cassette (SlUBIQ10 promoter region (SEQ ID NO: 23)/SlUBIQ10 terminator region (SEQ ID NO: 27)).

FIGS. 5A and 5B. Frequency of indels present in the plants of Table 3 at the target site of PSY1 gene (FIG. 5A) and CRTiso gene (FIG. 5B) (gRNA—guide RNA; psy—PSY1 gene; ubi10—SlPOLYUBIQUITIN10 cassette (SlUBIQ10 promoter region (SEQ ID NO: 23)/SlUBIQ10 terminator region (SEQ ID NO: 27)); #—identifies the different tomato lines: rep—BeYDV Replicon; ubi4—PcUBIQUITIN4-2 promoter; crtiso—CRTiso gene).

FIG. 6. Constructs used for gene targeting optimization. Two different constitutive promoters for the expression of the Cas9 endonuclease were used, PcUbiquitin4-2 (Ubi4) and SlUbiquitin10 (Ubi10), with two different gRNAs, expressed under the U6-26 promoter, targeting either CRTISO or PSY1 genes. Each combination was tested with or without the Rep proteins expressed under the 35S promoter. The kanamycin resistance gene is indicated by the arrow “Nos:NPTII”. The geminiviral large intergenic regions (LIRs, stem loop structure) flanking the gRNA, the geminiviral short intergenic region (SIR, orange box) and the Cas9 to create a replicon that contains the CRISPR/Cas9 components. The T-DNA left and right borders (LB and RB) are shown as black boxes. Construct Ubi10, expressing only Cas9 and kanamycin resistance was used as negative control.

FIG. 7. Gene targeting constructs. The gene targeting construct (“TmicT1-donor-Rep-Ubi10” or “TmicT2-donor-Rep-Ubi10”) contains the CRISPR/Cas9 system and the Rep protein on the T-DNA outside the geminiviral replicon (defined by the LIR sequences) and the donor repair template is within the replicon. Two different gRNAs that were examined, target 1 (“T1”) and target 2 (“T2”) both specific to the deleted allele and not affecting the repaired allele. Control constructs have either the geminiviral replicon and gRNA with no Cas9 endonuclease (“TmicT1-donor-Rep”, “TmicT2-donor-Rep”) or only the CRISPR/Cas9 system and the geminiviral intergenic regions containing the donor repair template with no Rep proteins (“TmicT1-donor-Ubi10”, “TmicT2-donor-Ubi10”). Other controls have only the gRNA and the intergenic regions containing the donor repair template but with no Cas9 and Rep (“TmicT1-donor”, “TmicT2-donor”), or with only the Cas9 and Rep (“Rep-Ubi10”), or only Rep (“Rep”) or only Cas9 (“Ubi10”).

FIG. 8. Primers used for long range PCR and Sanger sequencing of gene targeting integration sites. Sanger sequencing using primers crtisoUpFw and delRv to verify the junction between the CRTISO locus and the 5′ homology arm (HA) of the donor repair template, and the junction between the 5′ HA of the donor and the 281 bp repair sequence, respectively. Primers delFw and 3′endFw were used to verify the junction between the 281 bp repair sequence and the 3′ HA of the donor, and the junction between the donor 3′ HA and the CRTISO locus. Sequencing results obtained had the expected sequences for all junctions.

FIG. 9. Primers used for detection of T₀ gene targeting plants zygosity. A scheme of the tangerine and WT repaired alleles with the position of the primers used for the screen. Primers crtiso2Fw and crtiso2Rv were used to check heterozygosity by differentiating a tangerine (668 bp) or WT (either if the donor repair template or a repaired allele, 949 bp) allele fragments, together with primers crtiso2Fw and crtiso4Rv that amplify a repair allele (2516 bp) and tangerine allele (2235 bp).

FIGS. 10A-10C. Gene targeting in Micro-Tom. Phenotypes of crtiso and psy1 mutants. FIG. 10A shows mutants with distinctive phenotypes in comparison to WT, from right to left: psy1 mutant yellow ripe fruit. crtiso mutant orange ripe fruit and WT red ripe fruit. FIG. 10B crtiso mutant plant (right) exhibiting late greening of the young leaves phenotype, two days after moving from tissue culture to soil, as compared to psy1 mutant plant with green young leaves phenotype (left). FIG. 10C WT yellow flower (right) in comparison to inflorescence of crtiso mutant plant with white petals and orange anthers (left).

FIGS. 11A-11B. Targeted mutagenesis in Micro-Tom. Sequence footprints of CRTISO and PSY1 genes. FIG. 11A shows sequence footprints in CRTISO as a result of non-homologous end-joining repair, and their frequencies in individual plants. Each column represents a different T₀ plant where the CRTISO gene has been targeted using different constructs, except for two plants with Ubi10:Cas9 and no gRNA (9 and 17, marked with asterisks) which served as a negative control. The color-code for the sequence of the indel footprints is shown on the right. The frequency of each footprint, determined from the frequency of Illumina reads, is shown on the X axis: footprints represented under 3% of the reads are grouped in “Others”. Fruit color phenotypes are represented right to the barplot of each plant. CRTISO target: gcgatgctaccagcattctgGGG. FIG. 11B shows sequence footprints in PSY1 as a result of non-homologous end-joining repair, and their frequencies in individual plants. Each column represents a different T₀ plant where the PSY1 gene has been targeted using different constructs, except for three plants with Ubi10:Cas9 and no gRNA (9, 17 and 19, marked with asterisks) which served as a negative control. The color-code for the sequence of the indel footprints is shown on the right. The frequency of each footprint, determined from the frequency of Illumina reads, is shown on the X axis; footprints represented under 3% of the reads are grouped in “Others”. Fruit color phenotypes are represented right to the barplot of each plant. PSY1 target: gagcgtatataatgctgcttTGG.

FIG. 12 shows the gene targeting strategy used for the experiments. Plants are transformed with a construct comprising Cas9 under the expression of the SlUbiquitin10 (Ubi10) promoter, a gRNA (U6-26:tangerine t1 or t2), the Rep protein, and a donor sequence comprising a truncated CRTISO WT coding region that deleted in the tangerine mutant.

FIG. 13 shows the design of CRISPR gRNAs tangerine. CRISPR gRNAs were designed to match target sequences (yellow boxes) in the tangerine allele that are destroyed upon insertion of repair sequence. Target 1 (“T1”, left) was designed so that it is destroyed upon repair of the gene by homologous recombination, as the CRISPR mandatory PAM sequence (light blue box) separates from the target sequence. Target 2 (“T2”, right) is destroyed because the deletion is in the middle of the target, and insertion of the 281 bp upon repair splits it.

FIG. 14 shows relative quantification (RQ) of replicons in plants transfected with Constructs with or without Rep. Three biological replicates of plants from 4 different gene targeting constructs described in FIG. 2. Results indicate up to ˜90 fold increase in copy number. Bars represent the standard error of biological replicates.

FIGS. 15A-15C show the validation of gene targeting with the TmicT2-donor-Rep-Ubi10 construct. FIG. 15A shows a scheme of the repaired WT allele with the position of the primers used for the analysis of integration events. FIGS. 15B and 15C show an EtBr-stained agarose gels of the PCR product using crtisoUpFw and delRv primers (2122 bp) (FIG. 15B) and using delFw and crtiso4R primers (1971 bp) (FIG. 15C).

FIGS. 16A and 16B show the detection of zygosity in T₀ plants with gene targeting phenotype. FIGS. 16A and 16B show electrophoresis in an EtBr agarose gel of PCR products obtained by amplification from both sides of the deletion with primers crtiso2Fw and crtiso2Rv (FIG. 16A) and primers crtiso2Fw and crtiso4Rv (FIG. 16B).

FIGS. 17A and 17B show Southern blot hybridization of 4 T₀ plants with gene targeting phenotype. FIG. 17A shows the location of the NsiI and SspI-HF restriction sites and of the probes (gray rectangles) used in the experiment. FIG. 17B shows the Southern blot results.

FIGS. 18A and 18B show EtBr-stained agarose gels of PCR amplifications of gene targeting integration sites at 5′ junction (FIG. 18A) and 3′ junction (FIG. 18B). PCR primers are shown in FIG. 8.

FIG. 19 shows Sanger sequencing of tangerine target sites. Target #1 and target #2 sequences are marked by brown and green boxes, respectively. Dashed lines show Cas9 cleavage site. Upper panel: not transformed tangerine mutant. Two middle panels: TmicT1-donor-Rep-Ubi10 transformed T₀ plants #3 and #1. Bottom panel: TmicT2-donor-Rep-Ubi10 transformed T₀ plant #2.

FIG. 20 shows a summary of the phenotypes obtained by gene targeting, gene targeting experiment and random mutagenesis. WT Micro-Tom tomato fruit that was used as the background for the gene targeting experiment shows a red glossy surface. Tangerine mutant fruit used as the background for the gene targeting assay transformations shows a yellow non-glossy (matte) surface. Crtiso CRISPR mutant is the result of gene targeting using the CRISPR/Cas9 system of the crtiso gene where the fruit surface remains glossy, as in the Micro-Tom WT fruit, but the color is yellow. A “repaired” tangerine mutant repaired by gene targeting obtaining a red tomato, with the non-glossy fruit surface.

DETAILED DESCRIPTION

The present subject matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that this disclosure is not limited to the specific products, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure.

A skilled artisan would appreciate that the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of disclosure herein. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

In some embodiments, the term “about”, refers to a deviance of between 0.0001-5% from the indicated number or range of numbers. In some embodiment, the term “about”, refers to a deviance of between 1-10% from the indicated number or range of numbers. In some embodiment, the term “about”, refers to a deviance of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, from the indicated number or range of numbers. In some embodiments, the term “about”, refers to a deviance of up to 25% from the indicated number or range of numbers.

In some embodiments, disclosed herein are methods for gene targeting in a plant cell comprising expressing a nuclease expressed under a ubiquitin regulatory sequence. A skilled artisan would appreciate that said methods allow the generation of genetically modified plants comprising a desirable phenotype.

Methods for Gene Targeting

Disclosed herein is a method for gene targeting in a plant cell, the method comprising: (a) introducing into said plant cell a first nucleic acid comprising a viral replicon comprising a donor nucleic acid sequence, said donor sequence targeted to a plant endogenous DNA sequence; (b) introducing into said plant cell a second nucleic acid comprising a nuclease system, wherein said nuclease system is targeted to said plant endogenous DNA sequence, and wherein at least one component of said nuclease system is expressed under a UBQ10 regulatory sequence; wherein homologous recombination occurs between the donor sequence and the plant endogenous sequence.

A skilled artisan would appreciate that gene targeting comprises a process by which the genetic information of a cell or an organism is changed. Similarly, in some embodiments gene targeting modifies the expression and/or activity of a gene of interest. In some embodiments, the term “gene targeting” can be used interchangeably with “site-directed mutagenesis”, “site-specific mutagenesis”, “oligonucleotide-directed mutagenesis”, “gene replacement”, “gene modification”, “knock-in”, “homology-directed repair”, “homology-dependent repair”, “gene targeting”, “targeted mutagenesis”, having all the same qualities and meanings.

In some embodiments, gene targeting comprises the introduction to a cell of a donor nucleic acid sequence, containing the desired mutation as well as at least one sequence complementary to the template DNA around the mutation site, so it can hybridize with the DNA in the gene of interest leading to homologous recombination. The mutation may be a single base change (a point mutation), multiple base changes, deletions, or insertions. In some embodiments, the donor sequence is then introduced or ectopically expressed into a host cell as a vector. Optionally, mutated cells can be selected by DNA sequencing to check that they contain the desired mutation.

In some embodiments, gene targeting comprises gene editing, gene replacement, or a combination of both.

A skilled artisan would appreciate that, in some embodiments, a viral replicon comprises an autonomously replication region of viral DNA that replicates from a single origin of replication. In some embodiments, nonessential regions of the viral genome are replaced by foreign sequences, in order for these foreign sequences to be expressed in transfected cells. In some embodiments, said foreign sequences comprise a donor nucleic acid sequence or a target gene. In some embodiments, said viral replicon is selected from a group comprising: a geminiviral replicon, a bean yellow dwarf virus (BeYDV) replicon, a cabbage leaf curl virus (CalCuV) replicon, a tomato leaf curl virus (ToLCV) replicon, a wheat dwarf virus (WDV) replicon, a tobacco rattle virus (TRV) replicon, or any combination thereof. In some embodiments, said viral replicon comprises a part of a naturally occurring viral replicon. In some embodiments, said viral replicon comprises essential parts of a naturally occurring viral replicon.

In some embodiments, a geminiviral replicon comprises 2 long intergenic regions (LIR), a short intergenic region (SIR), a replication initiator protein (Rep) or any combination thereof. Said 2 LIRs are present on the T-DNA. Following cleavage by Rep, the replicon comprises only one LIR.

In some embodiments, LIR and SIR are the only cis-elements required for replication of a geminiviral replicon. The LIR comprises a bi-directional promoter and a stem-loop structure that is needed for initiation of rolling-circle replication (RCR). The SIR is the origin of C-strand synthesis and contains transcription termination and polyadenylation signals. Rep is required for replication, and it can be supplied in trans, therefore it needs not be present in the viral replicon.

Methods for transforming a plant are known to those skilled in the art. One skilled in the art would appreciate that the terms “transformation” and “transforming” describe a process by which a foreign DNA, such as a recombinant nucleic acid molecule or an expression vector as described herein, enters and changes a recipient cell into a transformed, genetically altered or transgenic cell. Transformation may be stable, wherein the nucleic acid sequence is integrated into the organism genome and as such represents a stable and inherited trait, or transient, wherein the nucleic acid sequence is expressed by the cell transformed but is not integrated into the genome, and as such represents a transient trait. In some embodiments, a recombinant nucleic acid molecule described herein is stably transformed into the plant cell.

A skilled artisan would appreciate that a donor nucleic acid sequence might comprise any nucleic acid to be integrated into a plant cell. In some embodiments, said donor nucleic acid comprises at least one nucleotide region similar or identical to the plant genome. In some embodiments, said donor sequence is integrated into the cell genome by homologous recombination. In some embodiments, said donor sequence comprises a gene, a part of a gene, a regulatory sequence, or any combination thereof. In some embodiments, said donor nucleic acid sequence comprises a mutated gene, a mutated regulatory sequence, a mutated promoter, a mutated terminator, or any mutated version of a naturally occurring oligonucleotide sequence. In some embodiments, said naturally occurring oligonucleotide comprises a plant endogenous DNA sequence.

A skilled artisan would appreciate that a plant endogenous DNA sequence might comprise any endogenous DNA sequence that an artisan wishes to genetically modify. In some embodiments, said plant endogenous DNA sequence comprises a gene, a part of a gene, a regulatory sequence, or any combination thereof.

In some embodiments, a nuclease system comprises a system used for gene targeting. In some embodiments, a nuclease system is selected from a group comprising: a nickase, a zinc finger nuclease (ZFN) system, a transcription activator-like effector nuclease (TALEN) system, a meganuclease, or a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) nuclease, or any combination thereof.

A skilled artisan would appreciate that a nickase may comprise any enzyme that cuts one strand of a double-stranded DNA at a specific recognition nucleotide sequences, sometimes known as a restriction site. Such enzymes hydrolyse or cut only one strand of the DNA duplex, to produce DNA molecules that are “nicked”, rather than cleaved.

A skilled artisan would appreciate that the terms “zinc finger nuclease” or “ZFN” are interchangeable having all the same meanings and qualities, wherein a ZFN encompasses a chimeric protein molecule comprising at least one zinc finger DNA binding domain operatively linked to at least one nuclease capable of double-strand cleaving of DNA. In some embodiments, a ZFN system comprises a ZFN known in the art. In some embodiments, a ZFN system comprises a ZFN newly created to cleave a preselected site.

In some embodiments, a ZFN creates a double-stranded break at a preselected endogenous target site. In some embodiments, a ZFN comprises a DNA-binding domain and a DNA-cleavage domain, wherein the DNA binding domain is comprised of at least one zinc finger and is operatively linked to a DNA-cleavage domain. In another embodiment, a zinc finger DNA-binding domain is at the N-terminus of the chimeric protein molecule and the DNA-cleavage domain is located at the C-terminus of the molecule. In another embodiment, a zinc finger DNA-binding domain is at the C-terminus of the chimeric protein molecule and the DNA-cleavage domain is located at the N-terminus of the molecule. In another embodiment, a zinc finger binding domain encompasses the region in a zinc finger nuclease that is capable of binding to a target locus, for example a preselected endogenous target site as disclosed herein. In another embodiment, a zinc finger DNA-binding domain comprises a protein domain that binds to a preselected endogenous target site on at least one homologous chromosome. In another embodiment, a zinc finger DNA-binding domain comprises a protein domain that binds to a polymorphic allele on at least one homologous chromosome. In another embodiment, a zinc finger DNA-binding domain comprises a protein domain that binds to a preselected endogenous target site on both homologous chromosomes. In another embodiment, a zinc finger DNA-binding domain comprises a protein domain that binds to polymorphic alleles on both homologous chromosomes.

In some embodiments, a TALEN system comprises a TAL effector DNA binding domain and a DNA cleavage domain, wherein said TAL effector DNA binding domain binds within said preselected endogenous target site, thereby targeting the DNA cleavage domain to cleave the DNA within said preselected endogenous target site.

A skilled artisan would appreciate that the terms “transcription activator-like effector nuclease”, “TALEN”, and “TAL effector nuclease” may be used interchangeably having all the same meanings and qualities, wherein a TALEN encompasses a nuclease capable of recognizing and cleaving its target site, for example a preselected endogenous target site as disclosed herein. In another embodiment, a TALEN comprises a fusion protein comprising a TALE domain and a nucleotide cleavage domain. In another embodiment, a TALE domain comprises a protein domain that binds to a nucleotide in a sequence-specific manner through one or more TALE-repeat modules. A skilled artisan would recognize that TALE-repeat modules comprise a variable number of about 34 amino acid repeats that recognize plant DNA sequences. Further, repeat modules can be rearranged according to a simple cipher to target new DNA sequences. In another embodiment, a TALE domain comprises a protein domain that binds to a preselected endogenous target site on at least one homologous chromosome. In another embodiment, a TALE domain comprises a protein domain that binds to a polymorphic allele on at least one homologous chromosome. In another embodiment, a TALE domain comprises a protein domain that binds to a preselected endogenous target site on both homologous chromosomes. In another embodiment, a TALE domain comprises a protein domain that binds to polymorphic alleles on both homologous chromosomes.

In one embodiment, a TALE domain comprises at least one of the TALE-repeat modules. In another embodiment, a TALE domain comprises from one to thirty TALE-repeat modules. In another embodiment, a TALE domain comprises more than thirty repeat modules. In another embodiment, a TALEN fusion protein comprises an N-terminal domain, one or more of TALE-repeat modules followed by a half-repeat module, a linker, and a nucleotide cleavage domain.

A skilled artisan would appreciate that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system comprises genome engineering tools based on the bacterial CRISPR/Cas prokaryotic adaptive immune system. This RNA-based technology is very specific and allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms (Belhaj K. et al., 2013. Plant Methods 2013, 9:39). In some embodiments, a CRISPR/Cas system comprises a CRISPR/Cas9 system.

In some embodiments, a CRISPR/Cas system comprises a Cas nuclease and a gRNA molecule, wherein said gRNA molecule binds within said preselected endogenous target site thereby guiding said Cas nuclease to cleave the DNA within said preselected endogenous target site.

A skilled artisan would appreciate that the terms “single-guide RNA”, “sgRNA”, and “gRNA” are interchangeable having all the same qualities and meanings. An sgRNA may encompass a chimeric RNA molecule which is composed of a targeting sequence (crRNA) and a Cas nuclease recruiting sequence (tracrRNA). In some embodiments, a crRNA is complementary to a genomic preselected target site, wherein the crRNA “targets” the CRISPR associated polypeptide (Cas) nuclease protein to the preselected target site.

In some embodiments, the length of crRNA sequence complementary is 19-22 nucleotides long e.g., 19-22 consecutive nucleotides complementary to the target site. In another embodiment, the length of crRNA sequence complementary to the region of DNA is about 15-30 nucleotides long. In another embodiment, the length of crRNA sequence complementary to the region of DNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In another embodiment, the length of crRNA sequence complementary to the region of DNA is 20 nucleotides long. In some embodiments, the crRNA is located at the 5′ end of the sgRNA molecule. In another embodiment, the crRNA comprises 100% complementation within the preselected target sequence. In another embodiment, the crRNA comprises at least 80% complementation within the preselected target sequence. In another embodiment, the crRNA comprises at least 85% complementation within the preselected target sequence. In another embodiment, the crRNA comprises at least 90% complementation within the preselected target sequence. In another embodiment, the crRNA comprises at least 95% complementation within the preselected target sequence. In another embodiment, the crRNA comprises at least 97% complementation within the preselected target sequence. In another embodiment, the crRNA comprises at least 99% complementation within the preselected target sequence. In another embodiment, a tracrRNA is 100-300 nucleotides long and provides a binding site for the Cas nuclease e.g., a Cas9 protein forming the CRISPR/Cas9 complex.

A skilled artisan would appreciate that a “target sequence” can be any DNA sequence that an artisan wishes to mutate. In some embodiments, the terms “target sequence” and “plant endogenous DNA sequence” are used herein interchangeably, having all the same qualities and meanings.

A skilled artisan would appreciate that a guide RNA may contain nucleotide sequences other than the region complementary or substantially complementary to a region of a target DNA sequence, for example a preselected endogenous target site. In another embodiment, a guide RNA comprises a crRNA or a derivative thereof. In another embodiment, a guide RNA comprises a crRNA: tracrRNA chimera.

In some embodiments, a CRISPR/Cas system comprises a single-guide RNA (sgRNA) and/or a Cas protein known in the art. In some embodiments, a CRISPR/Cas system comprises a single-guide RNA (sgRNA) and/or a Cas protein newly created to cleave at a preselected site.

In another embodiment, a CRISPR/Cas system comprises a Type I CRISPR-Cas system, or a Type II CRISPR-Cas system, or a Type III CRISPR-Cas system, or derivatives thereof. In another embodiment, a CRISPR-Cas system comprises an engineered and/or programmed nuclease system derived from naturally accruing CRISPR-Cas systems. In another embodiment, a CRISPR-Cas system comprises engineered and/or mutated Cas proteins. In another embodiment, a CRISPR-Cas system comprises engineered and/or programmed guide RNA.

In another embodiment, a gRNA molecule comprises a domain that is complementary to and binds to a preselected endogenous target site on at least one homologous chromosome. In another embodiment, a gRNA molecule comprises a domain that is complementary to and binds to a polymorphic allele on at least one homologous chromosome. In another embodiment, a gRNA molecule comprises a domain that is complementary to and binds to a preselected endogenous target site on both homologous chromosomes. In another embodiment, a gRNA molecule comprises a domain that is complementary to and binds to polymorphic alleles on both homologous chromosomes.

Cas enzymes comprise RNA-guided DNA endonuclease able to make double-stranded breaks (DSB) in DNA. The term “Cas enzyme” may be used interchangeably with the terms “CRISPR-associated endonucleases” or “CRISPR-associated polypeptides” having all the same qualities and meanings. In one embodiment, a Cas enzyme is selected from the group comprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, C2c1, CasX, NgAgo, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4, or homologs thereof, or modified versions thereof. In another embodiment, a Cas enzyme comprises Cas9. In another embodiment, a Cas enzyme comprises Cas1. In another embodiment, a Cas enzyme comprises Cas1B. In another embodiment, a Cas enzyme comprises Cas2. In another embodiment, a Cas enzyme comprises Cas3. In another embodiment, a Cas enzyme comprises Cas4. In another embodiment, a Cas enzyme comprises Cas5. In another embodiment, a Cas enzyme comprises Cas6. In another embodiment, a Cas enzyme comprises Cas7. In another embodiment, a Cas enzyme comprises Cas8. In another embodiment, a Cas enzyme comprises Cas10. In another embodiment, a Cas enzyme comprises Cpf1. In another embodiment, a Cas enzyme comprises Csy1. In another embodiment, a Cas enzyme comprises Csy2. In another embodiment, a Cas enzyme comprises Csy3. In another embodiment, a Cas enzyme comprises Cse1. In another embodiment, a Cas enzyme comprises Cse2. In another embodiment, a Cas enzyme comprises Csc1. In another embodiment, a Cas enzyme comprises Csc2. In another embodiment, a Cas enzyme comprises Csa5. In another embodiment, a Cas enzyme comprises Csn2. In another embodiment, a Cas enzyme comprises Csm2. In another embodiment, a Cas enzyme comprises Csm3. In another embodiment, a Cas enzyme comprises Csm4. In another embodiment, a Cas enzyme comprises Csm5. In another embodiment, a Cas enzyme comprises Csm6. In another embodiment, a Cas enzyme comprises Cmr1. In another embodiment, a Cas enzyme comprises Cmr3. In another embodiment, a Cas enzyme comprises Cmr4. In another embodiment, a Cas enzyme comprises Cmr5. In another embodiment, a Cas enzyme comprises Cmr6. In another embodiment, a Cas enzyme comprises Csb1. In another embodiment, a Cas enzyme comprises Csb2. In another embodiment, a Cas enzyme comprises Csb3. In another embodiment, a Cas enzyme comprises Csx17. In another embodiment, a Cas enzyme comprises Csx14. In another embodiment, a Cas enzyme comprises Csx10. In another embodiment, a Cas enzyme comprises Csx16, CsaX. In another embodiment, a Cas enzyme comprises Csx3. In another embodiment, a Cas enzyme comprises Csx1, Csx15, Csf1. In another embodiment, a Cas enzyme comprises Csf2. In another embodiment, a Cas enzyme comprises Csf3. In another embodiment, a Cas enzyme comprises Csf4. In another embodiment, a Cas enzyme comprises Cpf1. In another embodiment, a Cas enzyme comprises C2c1. In another embodiment, a Cas enzyme comprises CasX. In another embodiment, a Cas enzyme comprises NgAgo. In another embodiment, a Cas enzyme is Cas homologue. In another embodiment, a Cas enzyme is a Cas orthologue. In another embodiment, a Cas enzyme is a modified Cas enzyme. In another embodiment, a Cas enzyme is any CRISPR-associated endonucleases known in the art.

In some embodiment, a nuclease gene of the nuclease system is operably linked to a gene enhancer. In some embodiments, a nuclease gene of the nuclease system is operably linked to an UBQ10 regulatory sequence. In some embodiments, a Cas nuclease gene is operably linked to an UBQ10 regulatory sequence. In some embodiment, a gRNA of the nuclease system is operably linked to a gene enhancer. In some embodiments, a gRNA of the nuclease system is operably linked to an UBQ10 regulatory sequence.

In some embodiment, a nuclease gene and a gRNA are operably linked to a gene enhancer. In some embodiments, a nuclease gene and a gRNA are operably linked to an UBQ10 regulatory sequence. In some embodiments, a Cas nuclease gene and a gRNA are operably linked to an UBQ10 regulatory sequence.

In some embodiments, disclosed herein is a method for gene targeting in a plant cell, wherein no selection marker or reporter gene are used for generating said transgenic plant cell.

In some embodiments, a nuclease system is ectopically expressed in a plant cell by introducing into said plant cell a nucleic acid encoding said nuclease system. In some embodiments, a single expression vector comprises a nucleotide encoding a nuclease system, a viral replicon and a donor sequence.

There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (See Potrykus I 1991. Annu Rev Plant Physiol Plant Mol Biol 42, 205-225; Shimamoto K. et al., 1989. Nature 338, 274-276). Transformation methods may include, for example, but are not limited to, the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses and microprojection.

Plant transformation methods are fully described in U.S. Patent Application Publications US 20110209247: US 20110113514: US 20100199371; US 20070079396; US 20080307541; US 20030028913: and US20030196219; and U.S. Pat. No. 5,015,580: U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 6,160,208; U.S. Pat. No. 6,399,861; U.S. Pat. No. 6,403,865; U.S. Pat. No. 5,635,055; U.S. Pat. No. 5,824,877; U.S. Pat. No. 5,591,616; U.S. Pat. No. 5,981,840 and U.S. Pat. No. 6,384,301, which are incorporated by reference herein in their entirety.

In some embodiments, the transformation can be performed by an Agrobacterium-mediated gene transfer. The Agrobacterium-mediated system includes the use of plasmid vectors that contain defined DNA segments which integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. The transformation can be performed with any suitable tissue explant that provides a good source for initiation of whole-plant differentiation (See Horsch et al., 1988. Plant Molecular Biology Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht).

In some embodiments, the transformation can be performed by a viral vector-based gene transfer. In some embodiments, the transformation can be performed with the use of a virus vector to generate, for example, plants expressing a nuclease system, a donor nucleic acid sequence, or a target gene. In some embodiments, the transformation can be performed with the use of a virus vector to generate. for example, plants having an increased rate of mutagenesis. In some embodiments, the transformation can be performed with the use of a virus vector to generate, for example, plants having an increased rate of gene targeting. In some embodiments, the transformation can be performed with the use of a virus vector to generate, for example, plants having an increased rate of targeted gene editing.

In some embodiments, the transformation can be performed by a direct DNA uptake method. There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field, opening up mini-pores to allow DNA to enter. In microinjection, the DNA is mechanically injected directly into the cells using micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

In some embodiments, gene targeting is used for enhancing a beneficial trait or quality in a plant. In some embodiments, gene targeting is used for reducing a detrimental trait or quality in a plant. In some embodiments, gene targeting is used for enabling a plant to adapt to changing environmental conditions.

Expression of a Gene Linked to a UBQ10 Regulatory Sequence

In some embodiments, disclosed herein is a recombinant nucleic acid molecule comprising a first nucleotide sequence encoding a gene of interest, wherein said gene of interest is operably linked to a regulatory sequence of the gene encoding Ubiquitin10 (UBQ10).

A skilled artisan would appreciate that the term “operably linked” may encompass a functional linkage between two nucleic acid sequences. For example, a promoter sequence can be operably linked to a sequence encoding a gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest. A skilled artisan would appreciate that the terms “operably linked” and “fused” may in some embodiments be used interchangeably herein having the same meanings and qualities.

A skilled artisan would appreciate that a “regulatory sequence” comprises a segment of a nucleic acid molecule capable of increasing or decreasing the expression of genes operably linked to it. Further, the terms “regulatory sequence”, “regulatory element”, and “regulatory region” are used herein interchangeably having all the same qualities and meanings. In some embodiments, a regulatory sequence is selected from a group comprising: an enhancer, a silencer, a proximal promoter, a core promoter, an operator, a 5′ UTR, a 3′ UTR, or any combination thereof.

In some embodiments, a regulatory sequence comprises a part of an enhancer, a silencer, a proximal promoter, a core promoter, an operator, a 5′ UTR, a 3′ UTR, or any combination thereof. In some embodiments, a regulatory sequence comprises a promoter-terminator cassette. In some embodiments a gene of interest can be inserted adjacently to a regulatory sequence. In some embodiments a gene of interest can be inserted within a regulatory sequence. In some embodiments, a regulatory sequence comprises a contiguous chromosomal region. In some embodiments a regulatory sequence comprises a non-continuous chromosomal region.

In some embodiments, a region adjacent to a gene of interest comprises a regulatory sequence that regulates its expression. In some embodiments, the terms “regulatory sequence” and “adjacent region” are used interchangeably having all the same qualities and meanings.

A skilled artisan would appreciate that the term “promoter” or “promoter region” may encompass a nucleic acid control sequence located upstream from the transcriptional start of a gene, and which is involved in recognizing and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid sequence encoding a target protein.

Plant promoters are not well defined, and it is common to use a region located 1-2 Kb upstream of the start codon of a plant with the hope of including the plant gene promoter region. This upstream region, which includes the 5′ UTR, may or may not include all of the promoter. Further, it cannot be ruled out that an enhancer of the gene is located even 5 Kb upstream or elsewhere in the genome. Thus, even though upstream nucleic acid sequence of a gene is known, the exact location of the plant promoter may not be known.

In some embodiments, a promoter region comprises a 5′ UTR. In some embodiments, a promoter region comprises a 5′ UTR and additional upstream sequences. In some embodiments, a promoter comprises the entire region 1-2 Kb upstream of a start codon. In some embodiments, a promoter comprises the nucleic acid sequence about 1 Kb upstream of a start codon. In some embodiments, a promoter comprises the nucleic acid sequence about 2 Kb upstream of a start codon.

In some embodiments, a promoter region comprising a 5′ UTR is needed for the highest level of expression of a target gene. In some embodiments, a promoter region comprising a 5′ UTR and additional upstream sequences UTR is needed for the highest level of expression of a target gene. In some embodiments, a promoter comprising the entire region 1-2 Kb upstream of a start codon UTR is needed for the highest level of expression of a target gene. In some embodiments, a promoter comprising the nucleic acid sequence about 1 Kb upstream of a start codon UTR is needed for the highest level of expression of a target gene. In some embodiments, a promoter comprising the nucleic acid sequence about 2 Kb upstream of a start codon UTR is needed for the highest level of expression of a target gene. In some embodiments, a promoter comprising the nucleic acid sequence more than 2 Kb upstream of a start codon UTR is needed for the highest level of expression of a target gene. In some embodiments, a promoter comprising part of the nucleic acid sequence about 2 Kb upstream of a start codon UTR is needed for the highest level of expression of a target gene.

In some embodiments, a “target gene” is a gene or a nucleic acid sequence that an artisan wishes to express in a cell. In some embodiments, a “target gene” and a “gene of interest” are used herein interchangeably having all the same qualities and meanings. In some embodiments, a “target gene” and a “donor sequence” are used herein interchangeably having all the same qualities and meanings.

In some embodiments, a regulatory region comprises regulatory elements, such as 5′ leaders and introns for enhancing transcription, 3′ untranslated regions (such as polyadenylation signals and sites), and DNA for transit or signal peptides.

In some embodiments, promoters functionally equivalent to UBQ10 promoters can be used. For the identification of functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter may be analyzed, for example, by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant. Suitable well-known reporter genes include, for example, but are not limited to, beta-glucuronidase or beta-galactosidase, green fluorescent protein (GFP). red-fluorescent protein (RFP), and luciferase. The promoter activity can be assayed, for example, by measuring the enzymatic activity. The promoter strength and/or expression pattern may then be compared to that of a reference promoter (such as the one used in the methods disclosed herein). Alternatively, promoter strength may be assayed by quantifying mRNA levels or by comparing mRNA levels of the nucleic acid used in the methods disclosed herein, with mRNA levels of housekeeping genes such as 18S rRNA, using methods known in the art, such as Northern blotting with densitometric analysis of autoradiograms, quantitative real-time PCR or RT-PCR (See Heid et al., 1996 Genome Methods 6: 986-994).

A skilled artisan would appreciate that the term “terminator” or “terminator region” may encompass a nucleic acid control sequence located downstream from the transcriptional stop of a gene, and which is involved in recognizing and binding of polypeptides that effectively stop transcription of a target protein.

In some embodiments, a terminator region comprises a 3′ UTR. In some embodiments, a terminator region comprises a 3′ UTR and additional downstream sequences. In some embodiments, a terminator comprises a region about 1 Kb downstream of a stop codon. In some embodiments, a terminator comprises an intron. In some embodiments, a terminator region comprising a 3′ UTR is needed for the highest efficiency stop of transcription.

A skilled artisan would appreciate that an enhancer is a short (50-1500 bp) region of DNA that can be bound by proteins to increase the likelihood that transcription of a particular gene will occur. These proteins are sometimes referred to as transcription factors. They can be located up to 1 Mbp (1,000,000 bp) away from the gene, upstream or downstream from the start site. Contrarily, silencers are oligonucleotide sequences capable of binding transcription regulation factors that inhibit the expression of an adjacent gene. These transcriptions factors are generally known as repressors.

UBQ10 Regulatory Elements

The terms “polyubiquitin10 gene”, “ubiquitin10 gene”, “ubi10”, and “UBQ10” are used herein interchangeably having all the same qualities and meanings. The genomic region located ˜2 kb upstream the UBQ10 contains a promoter region and the 5′ UTR. Notably, this 5′ UTR is conserved in plants. It is ˜400 to 600 bp long and contains a ˜300-500 bp long intron whose 3′ end is adjacent to the start codon of this gene. The presence of this intron allows high gene expression in eukaryotes, especially when expressing prokaryotic genes, as exon-exon junctions are known to enhance gene expression in eukaryotes.

In some embodiments, wherein the UBQ10 is from tomato or potato, the 5′ UTR is 593 bp long and contains a 494 bp long intron. In some embodiments, wherein the UBQ10 is from Arabidopsis, the 5′ UTR is 389 bp long with an intron of 304 bp.

In some embodiments, the UBQ10 comprises a plant gene. In some embodiments, the UBQ10 comprises a Solanaceous plant gene. In some embodiments, the Solanaceous plant is selected from the group comprising a cultivated tomato plant, a wild-tomato plant, a cultivated potato plant, a wild-potato plant, an aubergine plant, a chili pepper plant, and a bell pepper plant. A skilled artisan would appreciate that the Solanaceous family comprises a large number species (over 3,000), including but not limited to species of tomatoes, peppers, chilies, aubergine, potatoes, and tobacco plants. In some embodiments, the UBQ10 is selected from any species of Solanaeceous plant.

In some embodiments, the UBQ10 comprises an Arabidopsis plant gene. In some embodiments, the UBQ10 comprises an Arabidopsis thaliana plant gene.

The nucleic acid sequences of UBQ10s are well known in the art and publicly available in genetic sequence databases. In some embodiments, the POLYUBIQUITIN10 nucleic acid gene sequences of tomato, potato, and Arabidopsis comprise SEQ ID Nos: 1, 3, and 5, respectively, wherein the encoded amino acid sequences of tomato, potato, and Arabidopsis are SEQ ID Nos: 2, 4, and 6, respectively.

The Polyubiquitin 10 protein encoded by the UBQ10 is involved in protein degradation. The Polyubiquitin 10 polypeptide consists of six polypeptide repeats. It is located on the chromosome 7 in tomato (Solyc07g064130) and potato (Sotub07g026130).

In some embodiments, a nucleic acid sequence comprising a tomato UBQ10 comprises the following nucleic acid sequence:

(SEQ ID NO: 22) gtcaactaccccaatttaaattttatttgattaagatatttttatggacc tactttataattaaaaatattttctatttgaaaaggaaggacaaaaatca tacaattttggtccaactactcctctctttttttttttggctttataaaa aaggaaagtgattagtaataaataattaaataatgaaaaaaggaggaaat aaaattttcgaattaaaatgtaaaagagaaaaaggagagggagtaatcat tgtttaactttatctaaagtaccccaattcgattttacatgtatatcaaa ttatacaaatattttattaaaatatagatattgaataattttattattct tgaacatgtaaataaaaattatctattatttcaatttttatataaactat tatttgaaatctcaattatgattttttaatatcactttctatccatgata atttcagcttaaaaagttttgtcaataattacattaattttgttgatgag gatgacaagatttcggtcatcaattacatatacacaaattgaaatagtaa gcaacttgattttttttctcataatgataatgacaaagacacgaaaagac aattcaatattcacattgatttatttttatatgataataattacaataat aatattcttataaagaaagagatcaattttgactgatccaaaaatttatt tatttttactataccaacgtcactaattatatctaataatgtaaaacaat tcaatcttacttaaatattaatttgaaataaactatttttataacgaaat tactaaatttatccaataacaaaaaggtcttaagaagacataaattcttt ttttgtaatgctcaaataaatttgagtaaaaaagaatgaaattgagtgat ttttttttaatcataagaaaataaataattaatttcaatataataaaaca gtaatataatttcataaatggaattcaatacttacctcttagatataaaa aataaatataaaaataaagtgtttctaataaacccgcaatttaaataaaa tatttaatattttcaatcaaatttaaataattatattaaaatatcgtaga aaaagagcaatatataatacaagaaagaagatttaagtacaattatcaac tattattatactctaattttgttatatttaatttcttacggttaaggtca tgttcacgataaactcaaaatacgctgtatgaggacatattttaaatttt aaccaataataaaactaagttatttttagtatatttttttgtttaacgtg acttaatttttcttttctagaggagcgtgtaagtgtcaacctcattctcc taattttcccaaccacataaaaaaaaaataaaggtagcttttgcgtgttg atttggtacactacacgtcattattacacgtgttttcgtatgattggtta atccatgaggcggtttcctctagagtcggccataccatctataaaataaa gctttctgcagctcattttttcatcttctatctgatttctaattataatt tctctgaattgccttcaaattctctttcaaggttagaatttttctctatt tttggtttttgtttgtttagattctgagtttagttaatcaggtgctgtta aagccctaaattttgagttttttcggttgttttgatggaaaatacctaac aattgagttttttcatgttgttttgtcggagaatgcctacaattggagtt cctttcgttgttttgatgagaaagcccctaatttgagtgtttttccgtcg atttgattttaaaggtttatattcgagtttttttcgtcggtttaatgaga aggcctaaaataggagtttttctggttgatttgactaaaaaagccatgga attttgtgtttttgatgtcgctttggttctcaaggcctaagatctgagtt tctccggttgttttgatgaaaaagccctaaaattggagtttttatcttgt gttttaggttgttttaatccttataatttgagttttttcgttgttctgat tgttgtttttatgaattttgcagATGCAGATCTTTGTGAAAACTCTCACC GGAAAGACTATCACCCTAGAGGTGGAAAGTTCTGATACAATCGACAACGT TAAGGCTAAGATCCAGGATAAGGAAGGAATTCCCCCGGATCAGCAAAGGC TTATCTTCGCTGGAAAGCAGTTGGAGGACGGACGTACTCTAGCTGATTAC AACATCCAGAAGGAGTCCACCCTCCATTTGGTGCTCCGTCTACGTGGTGG TATGCAGATCTTCGTGAAGACTCTCACGGGTAAGACGATTACCCTTGAGG TCGAAAGCTCAGACACCATTGACAACGTCAAGGCTAAGATCCAGGATAAG GAAGGCATTCCCCCAGACCAGCAGAGGTTGATCTTTGCAGGAAAGCAGTT GGAAGATGGCCGCACCCTAGCTGACTACAACATCCAGAAGGAGTCCACCC TCCATTTGGTGCTCCGTCTCCGTGGTGGTATGCAGATCTTCGTTAAGACT CTTACCGGAAAGACCATCACTTTGGAGGTGGAAAGCTCCGACACCATTGA CAACGTGAAGGCTAAGATCCAGGATAAGGAAGGGATCCCCCCAGACCAGC AGAGGTTGATCTTCGCTGGAAAGCAGCTCGAGGATGGTCGCACCCTGGCT GACTACAACATCCAGAAGGAGTCTACCCTCCATCTTGTCCTCCGTCTCCG TGGTGGTATGCAGATTTTTGTTAAGACCCTCACCGGAAAGACCATCACTT TGGAGGTGGAAAGCTCCGACACCATTGATAATGTTAAGGCTAAGATCCAG GACAAGGAGGGAATTCCTTCAGACCAGCAGAGGTTGATTTTCGCTGGTAA GCAGCTCGAGGACGGCCGCACCCTTGCCGACTACAACATCCAGAAGGAGT CGACCCTTCACCTTGTCCTCCGTCTACGTGGTGGTATGCAAATCTTTGTG AAGACCCTTACCGGGAAAACCATCACCCTGGAGGTTGAGAGCTCCGACAC CATTGACAATGTCAAGGCCAAGATCCAAGACAAGGAGGGTATTCCCCCAG ACCAGCAGAGGTTGATTTTTGCTGGCAAGCAGCTCGAGGATGGCCGCACT TTGGCGGACTATAACATCCAAAAGGAGTCGACCCTGCACTTGGTGCTTAG GCTGAGGGGAGGAATGCAGATCTTTGTGAAGACCTTGACCGGGAAGACCA TCACTTTGGAGGTGGAGAGTTCTGACACCATCGACAATGTGAAAGCTAAG ATTCAGGACAAGGAGGGGATCCCACCAGACCAGCAGAGGTTGATTTTCGC TGGTAAGCAGCTTGAGGATGGCCGCACCCTTGCTGACTACAATATCCAGA AGGAGTCCACCCTGCACCTTGTCCTCCGTCTCCGTGGTGGTTTTTAAgtt gtggttgtctggttgcgtctgttgcccgttgtctgttgcccattgtggtg gttgtgtttgtatgatggtcgttaaggatcatcaatgtgttttcgctttt tgttccattctgtttctcatttgtgaataataatggtatctttatgaata tgcagtttgtggtttcttttctgAttgcagttctgagcattttgtttttg cttccgtttactataccacttacagtttgcactaatttagttgatatgcg agccatctgatgtttgatgattcaaatggcgtttatgtaactcgtacccg agtggatggagaagagctccattgccggtttgtttcatgggtggcggagg gcaactcctgggaaggaacaaaagaaaaaccgtgatacgagttcatgggt gagagctccagcttgatcccttctctgtcgatcaaatttgaatttttgga tcacggcaggctcacaagataatccaaagtaaaacataatgaatagtact tctcaatgatcacttatttttagcaaatcagcaattgtgcatgtcaaatg atttcggtgtaagagaaagagttgatgaatcaaaatatctgtagctggat caagaatctgaggcagttgtatgtatcaatgatctttccgctacaatgat gttagctatccgagtcaaattgttgtagaattgcatacttcggcatcaca ttctggatgacataataaataggaagtcttcagatccctaaaaaattgag agctaataacattagtcctagatgtaactgggtgacaaccaagaaagaga catgcaaatactacttttgtttgaaggagcatccctggtttgacatattt tttctgaatatcaaactttgaaactctacctagtctaatgtctaacgaca gatcttactggtttaactgcagtgatatctactatcttttggaatgtttt ctccttcagttatacatcaagttccaagatgcaggtgtgcttgattgatg tacatggctgtgagaagtgcatcctgatgttcagatgatggttcattcta atgtcttttccttcaatcagttttctcagtctgacttagcttgtttcatc tgcatgtttgaatgttcgtttactcatagtaattgcatttttgtagcaga acatatcattggtcatggtttcaactgtgcgcgagtcttatgcttattca aactaggaaagcctccgtctagagggtacacgagttgttgctctgtgtgc gtcagtccatagtattaatcttgctagttgtagtatattgtttatgtgga ctcggaattcatcatatgctccttctttgcatcaagtaaggcaaggtaat gtatagaagctttttaactctttcatggaagctggcctttgccagcatac catccagaagatatcaaccctgcatcttggctgccg

The coding region of the UBQ10 of SEQ ID NO: 22 is shown in all capital letters. The ATG start codon for transcription of the POLYUBIQUTIN10 gene (SEQ ID NO: 22) is shown in bold (nucleotides 276-278 of SEQ ID NO: 22). The TAA stop codon for the stop of transcription of the POLYUBIQUTIN10 gene (SEQ ID NO: 22) is shown in bold (nucleotides 3447-3449 of SEQ ID NO: 22). In one embodiment, the polynucleotide sequence of a tomato UBQ10 comprises a homolog of SEQ ID NO: 22. In one embodiment, the polynucleotide sequence of a tomato UBQ10 is at least 70% homologous to SEQ ID NO: 22. In another embodiment, the polynucleotide sequence of a tomato UBQ10 is at least 80% homologous to SEQ ID NO: 22. In another embodiment, the polynucleotide sequence of a tomato UBQ10 is at least 90% homologous to SEQ ID NO: 22. In another embodiment, the polynucleotide sequence of a tomato UBQ10 is at least 95% homologous to SEQ ID NO: 22. In another embodiment, the polynucleotide sequence of a tomato UBQ10 is at least 98% homologous to SEQ ID NO: 22. In another embodiment, the polynucleotide sequence of a tomato UBQ10 is at least 99% homologous to SEQ ID NO: 22. A skilled artisan would appreciate the homology may be determined using BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters.

In some embodiments, a nucleic acid sequence comprising a potato UBQ10 comprises the following nucleic acid sequence:

(SEQ ID NO: 33) ccaagacaatttcagcttaaaaagttttattaatatttacattagttttg ttgatgaggatgacaagattttggtcatcaattacatatacccaaattga atacttagtaagcaacttaatgtttttcataatgataatgacagacacaa aaaaaacccatttattattcacattgattgatttttatatgcactatagt aataataataatatttcttataaagcaagaggtcaattttttattttatt ataccaacgacactaaattatatttgataatgtaaaacaattcaatttta cttaaatatcatgaaataaactatttttataaccaaattactaaatttat ccaataaaaaaaagtcattaagaagacataaaataaatttgagtaaaaag agtgaagtcgactgacttttttttttatcataagaaaataaattattaac tttaacctaataaaacactaatataatttcatggaatctaatacttacct cttagatataagaaaaagcgtttctaatagaccctcaatttacattaaat attttcaatcaagtttaaataacaaatatcaatatgaggtcaataacagt atcaaaataatatgaaaaaagagcaatacataatataagaaagaagattt aagtgcacttatcaaggtagtattatatcctaatttgctaatatttaaac tcttatatttaaggtcatgttcacgataaacttgaaatgcgctttattag agcatatattaaaataaaaaaaatacctaaaataaaataaagttattttt agtatatattttttacatgacctacatttttctagttttttctaaaggag cgtgtaagtgtcaacctcattctcctaattttccccaccacataaaaatt aaaaaggaaaggtagcttttgcgtgttgttttggtacactacacctcatt attacacgtgtcctcatatagttggttaacccgtgaggcggtttcctcta gagtcggccatgccatctataaaatgaagctttctgcacctcaatttttc atcttctatctgatttctattataatttctattaattgccttcaaatttc tctttcaaggttagaaatcttctctattttttggtttttgtctgtttaga ttctcgaattagctaatcaggtgctgttaaagccctaaaatttgagtttt ttttccgtcgaattgatgctaaaggcttaaaattagagttttttcgtcgg tttgactctgaaggcctaaaatttggggttttccgggtgatttgatgata aagccctagaatttgagtttttttatttgtcggtttgatgaaaaaggcct taaatttaatttttttcccggttgatttgatgaaaaagccctagaatttg tgttttttcgtcggtttgattctaaaggcctaaaatttgagtttttccgg ttgttttgatgaaaaagccctaaaatttgagttttttccccgtgttttag attgtttggttttaattcttgaatcagataatcagggagtgtgaaaagcc ctaaaatttgagtttttttcgttgttctgattgttgtttttatgaatttg cagATGCAGATCTTTGTGAAAACTCTCACCGGAAAGACCATCACCCTAGA GGTGGAAAGTTCTGATACAATCGACAACGTTAAGGCTAAGATTCAGGATA AGGAAGGAATTCCCCCGGATCAGCAAAGGCTTATCTTCGCCGGAAAGCAG TTGGAGGACGGACGTACTCTAGCTGATTACAACATCCAGAAGGAGTCTAC CCTCCATTTGGTGCTCCGTCTACGTGGTGGTATGCAGATCTTCGTTAAGA CTCTTACGGGTAAGACGATTACCCTTGAGGTCGAAAGCTCAGACACCATT GACAATGTTAAGGCTAAGATCCAGGATAAGGAAGGCATTCCCCCAGACCA GCAGAGGTTGATCTTTGCAGGGAAACAGTTGGAAGATGGCCGCACCCTAG CTGACTACAACATCCAGAAGGAGTCTACCCTACATTTGGTCCTCCGTCTC CGTGGTGGTATGCAGATCTTCGTTAAGACTCTTACCGGAAAGACCATCAC TTTGGAGGTGGAAAGCTCCGACACCATTGACAACGTGAAGGCTAAGATCC AGGATAAGGAGGGAATTCCCCCAGACCAGCAGAGGTTGATCTTCGCTGGT AAGCAATTGGAGGACGGCCGCACCCTAGCTGACTACAACATCCAGAAGGA GTCTACCCTCCATCTTGTCCTCCGTCTCCGTGGTGGTATGCAGATTTTTG TTAAGACCCTCACCGGGAAGACCATCACTTTGGAGGTTGAAAGCTCCGAC ACCATTGATAATGTCAAGGCTAAGATCCAGGACAAGGAGGGAATTCCCCC AGACCAGCAGAGGTTGATCTTCGCTGGAAAGCAATTGGAGGATGGCCGCA CCCTAGCTGACTACAACATCCAGAAGGAGTCCACCCTTCACCTTGTCCTC CGTCTCCGTGGTGGTATGCAGATTTTTGTTAAGACCCTTACCGGGAAGAC CATCACCCTGGAGGTTGAGAGCTCCGACACCATTGACAATGTTAAGGCCA AGATCCAAGACAAGGAGGGTATTCCCCCAGACCAGCAGAGGTTGATCTTC GCTGGTAAACAGCTTGAGGATGGCCGCACCCTTGCGGACTACAACATTCA GAAGGAGTCCACCCTTCACTTGGTGCTGAGGCTGAGGGGAGGAATGCAGA TCTTTGTGAAGACCTTAACCGGGAAGACCATCACCTTGGAGGTGGAGAGT TCTGACACCATCGACAATGTGAAAGCTAAGATTCAGGACAAGGAGGGGAT CCCACCAGACCAGCAGAGGTTGATCTTTGCTGGTAAGCAGCTTGAAGATG GACGCACCCTTGCCGACTACAATATCCAGAAGGAGTCCACTCTGCACCTT GTCCTCCGTCTCCGTGGTGGTTTTTAAgttgcctgttgttggttgtcgtg ttgtctggctgtgtctgttgcccattgtggtggttatgtgtttgcattat ggtcttaaaggatcatcaatgtgttttcgctttctgttcctttctgtttc tcatttgtgaataataatggcgtctttatgaacatccaatttctggtttc ttttctgatcgcagtttgagtatttgtttttgcttttgcctccgtctatt acaccactttgcaattactataatatactaaaagccttcgatccatcttc tgtttgatgattcgaatggtatttatttaactcatacccaagtgaagcat aaagttagaggagagttcctgttccattgcctgtttgtatcatgagcaac tcatgttaataaacataagaaaaaccatgatgcaatctgtgtagctgata gactttgatgacagacgactcataagtaacaagagataacaaagaggaaa cataataaacatgtacgggaagtcctccaacaatgactataatcacatgt ttttgtagattagcaattgtacatgtcaaatgatcttggattaaggaagg agcttgtgaatcaaaacatctgaatttggacctagagtcttgaggtgatc gtactttggatggagagaccatgaataagaataaatgaatctggaactga gaactaaatggaagacacactgatccaacagattaagcttatgacattaa tcacagaaggtaactcggtgacaaccaagaacggggagctgcaaattcta ttgtcttaacaacggacctttactggtttaactgttatgatgtcttttat aggtggcttttgggttgttcttcgctctatccttttatgtaactttcaag aaccaaccaaatgcaggtgttctagatagatatacgtggcatgtgagaag ggaccctgaagttcagatgacggt.

The coding region of the UBQ10 of SEQ ID NO: 33 is shown in all capital letters. The ATG start codon for transcription of the POLYUBIQUTIN10 gene (SEQ ID NO: 33) is shown in bold (nucleotides 1604-1606 of SEQ ID NO: 33). The TAA stop codon for the stop of transcription of the POLYUBIQUTIN10 gene (SEQ ID NO: 33) is shown in bold (nucleotides 2975-2977 of SEQ ID NO: 33). In one embodiment, the polynucleotide sequence of a potato UBQ10 comprises a homolog of SEQ ID NO: 33. In one embodiment, the polynucleotide sequence of a potato UBQ10 is at least 70% homologous to SEQ ID NO: 33. In another embodiment, the polynucleotide sequence of a potato UBQ10 is at least 80% homologous to SEQ ID NO: 33. In another embodiment, the polynucleotide sequence of a potato UBQ10 is at least 90% homologous to SEQ ID NO: 33. In another embodiment, the polynucleotide sequence of a potato UBQ10 is at least 95% homologous to SEQ ID NO: 33. In another embodiment, the polynucleotide sequence of a potato UBQ10 is at least 98% homologous to SEQ ID NO: 33. In another embodiment, the polynucleotide sequence of a potato UBQ10 is at least 99% homologous to SEQ ID NO: 33.

In some embodiments, a nucleic acid sequence comprising an Arabidopsis UBQ10 comprises the following nucleic acid sequence:

(SEQ ID NO: 40) ctccacttctacttccagcacgcttcttacttttaccacagctcttgcac ctaaccataacaccttccctgtatgatcgcgaagcacccaccctaagcca cattttaatccttctgttggccatgccccatcaaagttgcacttaaccca agattgtggtggagcttcccatgtttctcgtctgtcccgacggtgttgtg gttggtgctttccttacattctgagcctctttccttctaatccactcatc tgcatcttcttgtgtccttactaatacctcattggttccaaattccctcc ctttaagcaccagctcgtttctgttcttccacagcctcccaagtatccaa gggactaaagcctccacattcttcagatcaggatattcttgtttaagatg ttgaactctatggaggtttgtatgaactgatgatctaggaccggataagt tcccttcttcatagcgaacttattcaaagaatgttttgtgtatcattctt gttacattgttattaatgaaaaaatattattggtcattggactgaacacg agtgttaaatatggaccaggccccaaataagatccattgatatatgaatt aaataacaagaataaatcgagtcaccaaaccacttgccttttttaacgag acttgttcaccaacttgatacaaaagtcattatcctatgcaaatcaataa tcatacaaaaatatccaataacactaaaaaattaaaagaaatggataatt tcacaatatgttatacgataaagaagttacttttccaagaaattcactga ttttataagcccacttgcattagataaatggcaaaaaaaaacaaaaagga aaagaaataaagcacgaagaattctagaaaatacgaaatacgcttcaatg cagtgggacccacggttcaattattgccaattttcagctccaccgtatat ttaaaaaataaaacgataatgctaaaaaaatataaatcgtaacgatcgtt aaatctcaacggctggatcttatgacgaccgttagaaattgtggttgtcg acgagtcagtaataaacggcgtcaaagtggttgcagccggcacacacgag tcgtgtttatcaactcaaagcacaaatacttttcctcaacctaaaaataa ggcaattagccaaaaacaactttgcgtgtaaacaacgctcaatacacgtg tcattttattattagctattgcttcaccgccttagctttctcgtgaccta gtcgtcctcgtcttttcttcttcttcttctataaaacaatacccaaagag ctcttcttcttcacaattcagatttcaatttctcaaaatcttaaaaactt tctctcaattctctctaccgtgatcaaggtaaatttctgtgttccttatt ctctcaaaatcttcgattttgttttcgttcgatcccaatttcgtatatgt tctttggtttagattctgttaatcttagatcgaagacgattttctgggtt tgatcgttagatatcatcttaattctcgattagggtttcatagatatcat ccgatttgttcaaataatttgagttttgtcgaataattactcttcgattt gtgatttctatctagatctggtgttagtttctagtttgtgcgatcgaatt tgtcgattaatctgagtttttctgattaacagATGCAGATCTTTGTTAAG ACTCTCACCGGAAAGACAATCACCCTCGAGGTGGAAAGCTCCGACACCAT CGACAACGTTAAGGCCAAGATCCAGGATAAGGAGGGCATTCCTCCGGATC AGCAGAGGCTTATTTTCGCCGGCAAGCAGCTAGAGGATGGCCGTACGTTG GCTGATTACAATATCCAGAAGGAATCCACCCTCCACTTGGTCCTCAGGCT CCGTGGTGGTATGCAGATTTTCGTTAAAACCCTAACGGGAAAGACGATTA CTCTTGAGGTGGAGAGTTCTGACACCATCGACAACGTCAAGGCCAAGATC CAAGACAAAGAGGGTATTCCTCCGGACCAGCAGAGGCTGATCTTCGCCGG AAAGCAGTTGGAGGATGGCAGAACTCTTGCTGACTACAATATCCAGAAGG AGTCCACCCTTCATCTTGTTCTCAGGCTCCGTGGTGGTATGCAGATTTTC GTTAAGACGTTGACTGGGAAAACTATCACTTTGGAGGTGGAGAGTTCTGA CACCATTGATAACGTGAAAGCCAAGATCCAAGACAAAGAGGGTATTCCTC CGGACCAGCAGAGATTGATCTTCGCCGGAAAACAACTTGAAGATGGCAGA ACTTTGGCCGACTACAACATTCAGAAGGAGTCCACACTCCACTTGGTCTT GCGTCTGCGTGGAGGTATGCAGATCTTCGTGAAGACTCTCACCGGAAAGA CCATCACTTTGGAGGTGGAGAGTTCTGACACCATTGATAACGTGAAAGCC AAGATCCAGGACAAAGAGGGTATCCCACCGGACCAGCAGAGATTGATCTT CGCCGGAAAGCAACTTGAAGATGGAAGAACTTTGGCTGACTACAACATTC AGAAGGAGTCCACACTTCACTTGGTCTTGCGTCTGCGTGGAGGTATGCAG ATCTTCGTGAAGACTCTCACCGGAAAGACTATCACTTTGGAGGTAGAGAG CTCTGACACCATTGACAACGTGAAGGCCAAGATCCAGGATAAGGAAGGAA TCCCTCCGGACCAGCAGAGGTTGATCTTTGCCGGAAAACAATTGGAGGAT GGTCGTACTTTGGCGGATTACAACATCCAGAAGGAGTCGACCCTTCACTT GGTGTTGCGTCTGCGTGGAGGTATGCAGATCTTCGTCAAGACTTTGACCG GAAAGACCATCACCCTTGAAGTGGAAAGCTCCGACACCATTGACAACGTC AAGGCCAAGATCCAGGACAAGGAAGGTATTCCTCCGGACCAGCAGCGTCT CATCTTCGCTGGAAAGCAGCTTGAGGATGGACGTACTTTGGCCGACTACA ACATCCAGAAGGAGTCTACTCTTCACTTGGTCCTGCGTCTTCGTGGTGGT TCTAAatctcgtctctgttatgcttaagaagttcaatgtttcgtttcatg taaaactttggtggtttgtgttttggggccttgtataatccctgatgaat aagtgttctactatgtttccgttcctgttatctctttctttctaatgaca agtcgaacttcttctttatcatcgcttcgtttttattatctgtgcttctt ttgtttaatacgcctgcaaagtgactcgactctgtttagtgcagttctgc gaaacttgtaaatagtccaattgttggcctctagtaatagatgtagcgaa agtgttgagctgttgggttctaaggatggcttgaacatgttaatctttta ggttctgagtatgatgaacattcgttgttgctaagaaatgcctgtaatgt cccacaaatgtagaaaatggttcgtacctttgtccaagcattgatatgtc tgatgagaggaaactgcaagatactgagcttggtttaacgaaggagaggc agtttcttccttccaaagcatttcatttgacaatgccttgatcatcttaa gtagagtttctgttgtggaaagtttgaaactttgaagaaacgactctcaa gtaaattgatgatcacaagtgaaagtgtatgttacataagtggatatttc accctttttccatcaatcaaaacatcatatagtaatccattggtttatac aaacatcaaaatacatttacctctgaaatgaggaaaaaaatgcaaagaga tttttgaaaatttccaacaaatg.

The coding region of the UBQ10 of SEQ ID NO: 40 is shown in all capital letters. The ATG start codon for transcription of the POLYUBIQUTIN10 gene (SEQ ID NO: 40) is shown in bold (nucleotides 1683-1685 of SEQ ID NO: 40). The TAA stop codon for the stop of transcription of the POLYUBIQUTIN10 gene (SEQ ID NO: 40) is shown in bold (nucleotides 3054-3056 of SEQ ID NO: 40). In one embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 comprises a homolog of SEQ ID NO: 40. In one embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 is at least 70% homologous to SEQ ID NO: 40. In another embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 is at least 80% homologous to SEQ ID NO: 40. In another embodiment, the polynucleotide sequence of a Arabidopsis UBQ10 is at least 90% homologous to SEQ ID NO: 40. In another embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 is at least 95% homologous to SEQ ID NO: 40. In another embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 is at least 98% homologous to SEQ ID NO: 40. In another embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 is at least 99% homologous to SEQ ID NO: 40.

In some embodiments, a nucleic acid sequence comprising an Arabidopsis UBQ10 comprises the following nucleic acid sequence:

(SEQ ID NO: 47) gtcgacgagtcagtaataaacggcgtcaaagtggttgcagccggcacaca cgagtcgtgtttatcaactcaaagcacaaatacttttcctcaacctaaaa ataaggcaattagccaaaaacaactttgcgtgtaaacaacgctcaataca cgtgtcattttattattagctattgcttcaccgccttagctttctcgtga cctagtcgtcctcgtcttttcttcttcttcttctataaaacaatacccaa agagctcttcttcttcacaattcagatttcaatttctcaaaatcttaaaa actttctctcaattctctctaccgtgatcaaggtaaatttctgtgttcct tattctctcaaaatcttcgattttgttttcgttcgatcccaatttcgtat atgttctttggtttagattctgttaatcttagatcgaagacgattttctg ggtttgatcgttagatatcatcttaattctcgattagggtttcatagata tcatccgatttgttcaaataatttgagttttgtcgaataattactcttcg atttgtgatttctatctagatctggtgttagtttctagtttgtgcgatcg aatttgtcgattaatctgagtttttctgattaacagATGCAGATCTTTGT TAAGACTCTCACCGGAAAGACAATCACCCTCGAGGTGGAAAGCTCCGACA CCATCGACAACGTTAAGGCCAAGATCCAGGATAAGGAGGGCATTCCTCCG GATCAGCAGAGGCTTATTTTCGCCGGCAAGCAGCTAGAGGATGGCCGTAC GTTGGCTGATTACAATATCCAGAAGGAATCCACCCTCCACTTGGTCCTCA GGCTCCGTGGTGGTATGCAGATTTTCGTTAAAACCCTAACGGGAAAGACG ATTACTCTTGAGGTGGAGAGTTCTGACACCATCGACAACGTCAAGGCCAA GATCCAAGACAAAGAGGGTATTCCTCCGGACCAGCAGAGGCTGATCTTCG CCGGAAAGCAGTTGGAGGATGGCAGAACTCTTGCTGACTACAATATCCAG AAGGAGTCCACCCTTCATCTTGTTCTCAGGCTCCGTGGTGGTATGCAGAT TTTCGTTAAGACGTTGACTGGGAAAACTATCACTTTGGAGGTGGAGAGTT CTGACACCATTGATAACGTGAAAGCCAAGATCCAAGACAAAGAGGGTATT CCTCCGGACCAGCAGAGATTGATCTTCGCCGGAAAACAACTTGAAGATGG CAGAACTTTGGCCGACTACAACATTCAGAAGGAGTCCACACTCCACTTGG TCTTGCGTCTGCGTGGAGGTATGCAGATCTTCGTGAAGACTCTCACCGGA AAGACCATCACTTTGGAGGTGGAGAGTTCTGACACCATTGATAACGTGAA AGCCAAGATCCAGGACAAAGAGGGTATCCCACCGGACCAGCAGAGATTGA TCTTCGCCGGAAAGCAACTTGAAGATGGAAGAACTTTGGCTGACTACAAC ATTCAGAAGGAGTCCACACTTCACTTGGTCTTGCGTCTGCGTGGAGGTAT GCAGATCTTCGTGAAGACTCTCACCGGAAAGACTATCACTTTGGAGGTAG AGAGCTCTGACACCATTGACAACGTGAAGGCCAAGATCCAGGATAAGGAA GGAATCCCTCCGGACCAGCAGAGGTTGATCTTTGCCGGAAAACAATTGGA GGATGGTCGTACTTTGGCGGATTACAACATCCAGAAGGAGTCGACCCTTC ACTTGGTGTTGCGTCTGCGTGGAGGTATGCAGATCTTCGTCAAGACTTTG ACCGGAAAGACCATCACCCTTGAAGTGGAAAGCTCCGACACCATTGACAA CGTCAAGGCCAAGATCCAGGACAAGGAAGGTATTCCTCCGGACCAGCAGC GTCTCATCTTCGCTGGAAAGCAGCTTGAGGATGGACGTACTTTGGCCGAC TACAACATCCAGAAGGAGTCTACTCTTCACTTGGTCCTGCGTCTTCGTGG TGGTTTCTAAatctcgtctctgttatgcttaagaagttcaatgtttcgtt tcatgtaaaactttggtggtttgtgttttggggccttgtataatccctga tgaataagtgttctactatgtttccgttcctgttatctctttctttctaa tgacaagtcgaacttcttctttatcatcgcttcgtttttattatctgtgc ttcttttgtttaatacgcctgcaaagtgactcgactctgtttagtgcagt tctgcgaaacttgtaaatagtccaattgttggcctctagtaatagatgta gcgaaagtgttgagctgttgggttctaaggatggcttgaacatgttaatc ttttaggttctgagtatgatgaacattcgttgttgctaagaaatgcctgt aatgtcccacaaatgtagaaaatggttcgtacctttgtccaagcattgat atgtctgatgagaggaaactgcaagatactgagcttggtttaacgaagga gaggcagtttcttccttccaaagcatttcatttgacaatgccttgatcat cttaagtagagtttctgttgtggaaagtttgaaactttgaagaaacgact ctcaagtaaattgatgatcacaagtgaaagtgtatgttacataagtggat atttcaccctttttccatcaatcaaaacatcatatagtaatccattggtt tatacaaacatcaaaatacatttacctctgaaatgaggaaaaaaatgcaa agagatttttgaaaatttccaacaaatg.

The coding region of the UBQ10 of SEQ ID NO: 47 is shown in all capital letters. The ATG start codon for transcription of the POLYUBIQUTIN10 gene (SEQ ID NO: 47) is shown in bold (nucleotides 637-639 of SEQ ID NO: 47). The TAA stop codon for the stop of transcription of the POLYUBIQUTIN10 gene (SEQ ID NO: 47) is shown in bold (nucleotides 2008-2010 of SEQ ID NO: 47). In one embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 comprises a homolog of SEQ ID NO: 47. In one embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 is at least 70% homologous to SEQ ID NO: 47. In another embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 is at least 80% homologous to SEQ ID NO: 47. In another embodiment, the polynucleotide sequence of a Arabidopsis UBQ10 is at least 90% homologous to SEQ ID NO: 47. In another embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 is at least 95% homologous to SEQ ID NO: 47. In another embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 is at least 98% homologous to SEQ ID NO: 47. In another embodiment, the polynucleotide sequence of an Arabidopsis UBQ10 is at least 99% homologous to SEQ ID NO: 47.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

gtaaatttctgtgttccttattctctcaaaatcttcgattttgttttcgt tcgatcccaatttcgtatatgttctttggtttagattctgttaatcttag atcgaagacgattttctgggtttgatcgttagatatcatcttaattctcg attagggtttcatagatatcatccgatttgttcaaataatttgagttttg tcgaataattactcttcgatttgtgatttctatctagatctggtgttagt ttctagtttgtgcgatcgaatttgtcgattaatctgagtttttctgatta acag

(SEQ ID NO: 44).

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 23) gtcaactaccccaatttaaattttatttgattaagatatttttatggacc tactttataattaaaaatattttctatttgaaaaggaaggacaaaaatca tacaattttggtccaactactcctctctttttttttttggctttataaaa aaggaaagtgattagtaataaataattaaataatgaaaaaaggaggaaat aaaattttcgaattaaaatgtaaaagagaaaaaggagagggagtaatcat tgtttaactttatctaaagtaccccaattcgattttacatgtatatcaaa ttatacaaatattttattaaaatatagatattgaataattttattattct tgaacatgtaaataaaaattatctattatttcaatttttatataaactat tatttgaaatctcaattatgattttttaatatcactttctatccatgata atttcagcttaaaaagttttgtcaataattacattaattttgttgatgag gatgacaagatttcggtcatcaattacatatacacaaattgaaatagtaa gcaacttgattttttttctcataatgataatgacaaagacacgaaaagac aattcaatattcacattgatttatttttatatgataataattacaataat aatattcttataaagaaagagatcaattttgactgatccaaaaatttatt tatttttactataccaacgtcactaattatatctaataatgtaaaacaat tcaatcttacttaaatattaatttgaaataaactatttttataacgaaat tactaaatttatccaataacaaaaaggtcttaagaagacataaattcttt ttttgtaatgctcaaataaatttgagtaaaaaagaatgaaattgagtgat ttttttttaatcataagaaaataaataattaatttcaatataataaaaca gtaatataatttcataaatggaattcaatacttacctcttagatataaaa aataaatataaaaataaagtgtttctaataaacccgcaatttaaataaaa tatttaatattttcaatcaaatttaaataattatattaaaatatcgtaga aaaagagcaatatataatacaagaaagaagatttaagtacaattatcaac tattattatactctaattttgttatatttaatttcttacggttaaggtca tgttcacgataaactcaaaatacgctgtatgaggacatattttaaatttt aaccaataataaaactaagttatttttagtatatttttttgtttaacgtg acttaatttttcttttctagaggagcgtgtaagtgtcaacctcattctcc taattttcccaaccacataaaaaaaaaataaaggtagcttttgcgtgttg atttggtacactacacgtcattattacacgtgttttcgtatgattggtta atccatgaggcggtttcctctagagtcggccataccatctataaaataaa gctttctgcagctcattttttcatcttctatctgatttctattataattt ctctgaattgccttcaaatttctctttcaaggttagaatttttctctatt ttttggtttttgtttgtttagattctgagtttagttaatcaggtgctgtt aaagccctaaattttgagtttttttcggttgttttgatggaaaataccta acaattgagttttttcatgttgttttgtcggagaatgcctacaattggag ttcctttcgttgttttgatgagaaagcccctaatttgagtgtttttccgt cgatttgattttaaaggtttatattcgagtttttttcgtcggtttaatga gaaggcctaaaataggagtttttctggttgatttgactaaaaaagccatg gaattttgtgtttttgatgtcgctttggttctcaaggcctaagatctgag tttctccggttgttttgatgaaaaagccctaaaattggagtttttatctt gtgttttaggttgttttaatccttataatttgagttttttcgttgttctg attgttgtttttatgaattttgcag.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 24) gtcaactaccccaatttaaattttatttgattaagatatttttatggacc tactttataattaaaaatattttctatttgaaaaggaaggacaaaaatca tacaattttggtccaactactcctctctttttttttttggctttataaaa aaggaaagtgattagtaataaataattaaataatgaaaaaaggaggaaat aaaattttcgaattaaaatgtaaaagagaaaaaggagagggagtaatcat tgtttaactttatctaaagtaccccaattcgattttacatgtatatcaaa ttatacaaatattttattaaaatatagatattgaataattttattattct tgaacatgtaaataaaaattatctattatttcaatttttatataaactat tatttgaaatctcaattatgattttttaatatcactttctatccatgata atttcagcttaaaaagttttgtcaataattacattaattttgttgatgag gatgacaagatttcggtcatcaattacatatacacaaattgaaatagtaa gcaacttgattttttttctcataatgataatgacaaagacacgaaaagac aattcaatattcacattgatttatttttatatgataataattacaataat aatattcttataaagaaagagatcaattttgactgatccaaaaatttatt tatttttactataccaacgtcactaattatatctaataatgtaaaacaat tcaatcttacttaaatattaatttgaaataaactatttttataacgaaat tactaaatttatccaataacaaaaaggtcttaagaagacataaattcttt ttttgtaatgctcaaataaatttgagtaaaaaagaatgaaattgagtgat ttttttttaatcataagaaaataaataattaatttcaatataataaaaca gtaatataatttcataaatggaattcaatacttacctcttagatataaaa aataaatataaaaataaagtgtttctaataaacccgcaatttaaataaaa tatttaatattttcaatcaaatttaaataattatattaaaatatcgtaga aaaagagcaatatataatacaagaaagaagatttaagtacaattatcaac tattattatactctaattttgttatatttaatttcttacggttaaggtca tgttcacgataaactcaaaatacgctgtatgaggacatattttaaatttt aaccaataataaaactaagttatttttagtatatttttttgtttaacgtg acttaatttttcttttctagaggagcgtgtaagtgtcaacctcattctcc taattttcccaaccacataaaaaaaaaataaaggtagcttttgcgtgttg atttggtacactacacgtcattattacacgtgttttcgtatgattggtta atccatgaggcggtttcctctagagtcggcca.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 25) taccatctataaaataaagctttctgcagctcattttttcatcttctatc tgatttctattataatttctctgaattgccttcaaatttctctttcaag.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 26) gttagaatttttctctattttttggtttttgtttgtttagattctgagtt tagttaatcaggtgctgttaaagccctaaattttgagtttttttcggttg ttttgatggaaaatacctaacaattgagttttttcatgttgttttgtcgg agaatgcctacaattggagttcctttcgttgttttgatgagaaagcccct aatttgagtgtttttccgtcgatttgattttaaaggtttatattcgagtt tttttcgtcggtttaatgagaaggcctaaaataggagtttttctggttga tttgactaaaaaagccatggaattttgtgtttttgatgtcgctttggttc tcaaggcctaagatctgagtttctccggttgttttgatgaaaaagcccta aaattggagtttttatcttgtgttttaggttgttttaatccttataattt gagttttttcgttgttctgattgttgtttttatgaattttgcag.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 34) ccaagacaatttcagcttaaaaagttttattaatatttacattagttttg ttgatgaggatgacaagattttggtcatcaattacatatacccaaattga atacttagtaagcaacttaatgtttttcataatgataatgacagacacaa aaaaaacccatttattattcacattgattgatttttatatgcactatagt aataataataatatttcttataaagcaagaggtcaattttttattttatt ataccaacgacactaaattatatttgataatgtaaaacaattcaatttta cttaaatatcatgaaataaactatttttataaccaaattactaaatttat ccaataaaaaaaagtcattaagaagacataaaataaatttgagtaaaaag agtgaagtcgactgacttttttttttatcataagaaaataaattattaac tttaacctaataaaacactaatataatttcatggaatctaatacttacct cttagatataagaaaaagcgtttctaatagaccctcaatttacattaaat attttcaatcaagtttaaataacaaatatcaatatgaggtcaataacagt atcaaaataatatgaaaaaagagcaatacataatataagaaagaagattt aagtgcacttatcaaggtagtattatatcctaatttgctaatatttaaac tcttatatttaaggtcatgttcacgataaacttgaaatgcgctttattag agcatatattaaaataaaaaaaatacctaaaataaaataaagttattttt agtatatattttttacatgacctacatttttctagttttttctaaaggag cgtgtaagtgtcaacctcattctcctaattttccccaccacataaaaatt aaaaaggaaaggtagcttttgcgtgttgttttggtacactacacctcatt attacacgtgtcctcatatagttggttaacccgtgaggcggtttcctcta gagtcggccaTGCCATCTATAAAATGAAGCTTTCTGCACCTCAATTTTTC ATCTTCTATCTGATTTCTATTATAATTTCTATTAATTGCCTTCAAATTTC TCTTTCAAGgttagaaatcttctctattttttggtttttgtctgtttaga ttctcgaattagctaatcaggtgctgttaaagccctaaaatttgagtttt ttttccgtcgaattgatgctaaaggcttaaaattagagttttttcgtcgg tttgactctgaaggcctaaaatttggggttttccgggtgatttgatgata aagccctagaatttgagtttttttatttgtcggtttgatgaaaaaggcct taaatttaatttttttcccggttgatttgatgaaaaagccctagaatttg tgttttttcgtcggtttgattctaaaggcctaaaatttgagtttttccgg ttgttttgatgaaaaagccctaaaatttgagttttttccccgtgttttag attgtttggttttaattcttgaatcagataatcagggagtgtgaaaagcc ctaaaatttgagtttttttcgttgttctgattgttgtttttatgaatttg cag.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 35) ccaagacaatttcagcttaaaaagttttattaatatttacattagttttg ttgatgaggatgacaagattttggtcatcaattacatatacccaaattga atacttagtaagcaacttaatgtttttcataatgataatgacagacacaa aaaaaacccatttattattcacattgattgatttttatatgcactatagt aataataataatatttcttataaagcaagaggtcaattttttattttatt ataccaacgacactaaattatatttgataatgtaaaacaattcaatttta cttaaatatcatgaaataaactatttttataaccaaattactaaatttat ccaataaaaaaaagtcattaagaagacataaaataaatttgagtaaaaag agtgaagtcgactgacttttttttttatcataagaaaataaattattaac tttaacctaataaaacactaatataatttcatggaatctaatacttacct cttagatataagaaaaagcgtttctaatagaccctcaatttacattaaat attttcaatcaagtttaaataacaaatatcaatatgaggtcaataacagt atcaaaataatatgaaaaaagagcaatacataatataagaaagaagattt aagtgcacttatcaaggtagtattatatcctaatttgctaatatttaaac tcttatatttaaggtcatgttcacgataaacttgaaatgcgctttattag agcatatattaaaataaaaaaaatacctaaaataaaataaagttattttt agtatatattttttacatgacctacatttttctagttttttctaaaggag cgtgtaagtgtcaacctcattctcctaattttccccaccacataaaaatt aaaaaggaaaggtagcttttgcgtgttgttttggtacactacacctcatt attacacgtgtcctcatatagttggttaacccgtgaggcggtttcctcta gagtcggcca.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 36) tgccatccataaaatgaagctttctgcacctcaatttttcatcttctatc tgatttccattataatttctattaattgccttcaaatttctctttcaag.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 37) gttagaaatcttctctattttttggtttttgtctgtttagattctcgaat tagctaatcaggtgctgttaaagccctaaaatttgagttttttttccgtc gaattgatgctaaaggcttaaaattagagttttttcgtcggtttgactct gaaggcctaaaatttggggttttccgggtgatttgatgataaagccctag aatttgagtttttttatttgtcggtttgatgaaaaaggccttaaatttaa tttttttcccggttgatttgatgaaaaagccctagaatttgtgttttttc gtcggtttgattctaaaggcctaaaatttgagtttttccggttgttttga tgaaaaagccctaaaatttgagttttttccccgtgttttagattgtttgg ttttaattcttgaatcagataatcagggagtgtgaaaagccctaaaattt gagtttttttcgttgttctgattgttgtttttatgaatttgcag.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 41) ctccacttctacttccagcacgcttcttacttttaccacagctcttgcac ctaaccataacaccttccctgtatgatcgcgaagcacccaccctaagcca cattttaatccttctgttggccatgccccatcaaagttgcacttaaccca agattgtggtggagcttcccatgtttctcgtctgtcccgacggtgttgtg gttggtgctttccttacattctgagcctctttccttctaatccactcatc tgcatcttcttgtgtccttactaatacctcattggttccaaattccctcc ctttaagcaccagctcgtttctgttcttccacagcctcccaagtatccaa gggactaaagcctccacattcttcagatcaggatattcttgtttaagatg ttgaactctatggaggtttgtatgaactgatgatctaggaccggataagt tcccttcttcatagcgaacttattcaaagaatgttttgtgtatcattctt gttacattgttattaatgaaaaaatattattggtcattggactgaacacg agtgttaaatatggaccaggccccaaataagatccattgatatatgaatt aaataacaagaataaatcgagtcaccaaaccacttgccttttttaacgag acttgttcaccaacttgatacaaaagtcattatcctatgcaaatcaataa tcatacaaaaatatccaataacactaaaaaattaaaagaaatggataatt tcacaatatgttatacgataaagaagttacttttccaagaaattcactga ttttataagcccacttgcattagataaatggcaaaaaaaaacaaaaagga aaagaaataaagcacgaagaattctagaaaatacgaaatacgcttcaatg cagtgggacccaggttcaattattgccaattttcagctccaccgtatatt taaaaaataaaacgataatgctaaaaaaatataaatcgtaacgatcgtta aatctcaacggctggatcttatgacgaccgttagaaattgtggttgtcga cgagtcagtaataaacggcgtcaaagtggttgcagccggcacacacgagt cgtgtttatcaactcaaagcacaaatacttttcctcaacctaaaaataag gcaattagccaaaaacaactttgcgtgtaaacaacgctcaatacacgtgt cattttattattagctattgcttcaccgccttagctttctcgtgacctag tcgtcctcgtcttttcttcttcttcttctataaaacaatacccaaagagc tcttcttcttcacaattcagatttcaatttctcaaaatcttaaaaacttt ctctcaattctctctaccgtgatcaaggtaaatttctgtgttccttattc tctcaaaatcttcgattttgttttcgttcgatcccaatttcgtatatgtt ctttggtttagattctgttaatcttagatcgaagacgattttctgggttt gatcgttagatatcatcttaattctcgattagggtttcatagatatcatc cgatttgttcaaataatttgagttttgtcgaataattactcttcgatttg tgatttctatctagatctggtgttagtttctagtttgtgcgatcgaattt gtcgattaatctgagtttttctgattaacag.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 42) ctccacttctacttccagcacgcttcttacttttaccacagctcttgcac ctaaccataacaccttccctgtatgatcgcgaagcacccaccctaagcca cattttaatccttctgttggccatgccccatcaaagttgcacttaaccca agattgtggtggagcttcccatgtttctcgtctgtcccgacggtgttgtg gttggtgctttccttacattctgagcctctttccttctaatccactcatc tgcatcttcttgtgtccttactaatacctcattggttccaaattccctcc ctttaagcaccagctcgtttctgttcttccacagcctcccaagtatccaa gggactaaagcctccacattcttcagatcaggatattcttgtttaagatg ttgaactctatggaggtttgtatgaactgatgatctaggaccggataagt tcccttcttcatagcgaacttattcaaagaatgttttgtgtatcattctt gttacattgttattaatgaaaaaatattattggtcattggactgaacacg agtgttaaatatggaccaggccccaaataagatccattgatatatgaatt aaataacaagaataaatcgagtcaccaaaccacttgccttttttaacgag acttgttcaccaacttgatacaaaagtcattatcctatgcaaatcaataa tcatacaaaaatatccaataacactaaaaaattaaaagaaatggataatt tcacaatatgttatacgataaagaagttacttttccaagaaattcactga ttttataagcccacttgcattagataaatggcaaaaaaaaacaaaaagga aaagaaataaagcacgaagaattctagaaaatacgaaatacgcttcaatg cagtgggacccacggttcaattattgccaattttcagctccaccgtatat ttaaaaaataaaacgataatgctaaaaaaatataaatcgtaacgatcgtt aaatctcaacggctggatcttatgacgaccgttagaaattgtggttgtcg acgagtcagtaataaacggcgtcaaagtggttgcagccggcacacacgag tcgtgtttatcaactcaaagcacaaatacttttcctcaacctaaaaataa ggcaattagccaaaaacaactttgcgtgtaaacaacgctcaatacacgtg tcattttattattagctattgcttcaccgccttagctttctcgtgaccta gtcgtcctcgtcttttcttcttcttcttctataaaacaatacc.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 43) caaagagctcttcttcttcacaattcagatttcaatttctcaaaatctta aaaactttctctcaattctctctaccgtgatcaag.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 48) gtcgacgagtcagtaataaacggcgtcaaagtggttgcagccggcacaca cgagtcgtgtttatcaactcaaagcacaaatacttttcctcaacctaaaa ataaggcaattagccaaaaacaactttgcgtgtaaacaacgctcaataca cgtgtcattttattattagctattgcttcaccgccttagctttctcgtga cctagtcgtcctcgtcttttcttcttcttcttctataaaacaatacccaa agagctcttcttcttcacaattcagatttcaatttctcaaaatcttaaaa actttctctcaattctctctaccgtgatcaaggtaaatttctgtgttcct tattctctcaaaatcttcgattttgttttcgttcgatcccaatttcgtat atgttctttggtttagattctgttaatcttagatcgaagacgattttctg ggtttgatcgttagatatcatcttaattctcgattagggtttcatagata tcatccgatttgttcaaataatttgagttttgtcgaataattactcttcg atttgtgatttctatctagatctggtgttagtttctagtttgtgcgatcg aatttgtcgattaatctgagtttttctgattaacag.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 49) gtcgacgagtcagtaataaacggcgtcaaagtggttgcagccggcacaca cgagtcgtgtttatcaactcaaagcacaaatacttttcctcaacctaaaa ataaggcaattagccaaaaacaactttgcgtgtaaacaacgctcaataca cgtgtcattttattattagctattgcttcaccgccttagctttctcgtga cctagtcgtcctcgtcttttcttcttcttcttctataaaacaatacc.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises a homolog of any one of SEQ ID NOs: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49. In one embodiment, a nucleic acid sequence comprising a first regulatory region is at least 70% homologous to any one of SEQ ID NOs: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49. In another embodiment, a nucleic acid sequence comprising a first regulatory region is at least 80% homologous to any one of SEQ ID NOs: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49. In another embodiment, a nucleic acid sequence comprising a first regulatory region is at least 90% homologous to any one of SEQ ID NOs: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49. In another embodiment, a nucleic acid sequence comprising a first regulatory region is at least 95% homologous to any one of SEQ ID NOs: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49. In another embodiment, a nucleic acid sequence comprising a first regulatory region is at least 98% homologous to any one of SEQ ID NOs: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49. In another embodiment, a nucleic acid sequence comprising a first regulatory region is at least 99% homologous to any one of SEQ ID NOs: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49.

In some embodiments, a nucleic acid sequence comprising a first regulatory region comprises a combination of any one of SEQ ID NOs: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49.

In some embodiments, an UBQ10 regulatory sequence comprises a Solanum lycopersicum UBQ10 regulatory sequence.

Expression of a Target Gene Linked to UBQ10

In some embodiments, disclosed herein is a recombinant nucleic acid molecule comprising: a first nucleic acid sequence of a first regulatory region; and a second nucleic acid sequence of a second regulatory region, wherein said first regulatory region is adjacent to the nucleic acid sequence encoding a UBQ10 extending about 2 Kb upstream but not including the start codon of the UBQ10, and wherein said second regulatory region is adjacent to the nucleic acid sequence encoding a UBQ10 extending about 1 Kb downstream from but not including the stop codon region of UBQ10, and wherein said first and second nucleic acid sequences are fused to a nucleic acid sequence of a target gene.

In some embodiments, a recombinant nucleic acid molecule disclosed herein comprises: a first nucleic acid sequence of a first regulatory region; and a second nucleic acid sequence of a second regulatory region, wherein said first region comprises a nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 2 Kb upstream but not including the start codon of the UBQ10, and wherein said second region comprises nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 1 Kb downstream from but not including the stop codon region of UBQ10, and wherein said first and second nucleic acid sequences are operably linked to a nucleic acid sequence of a target gene, wherein the first nucleic acid sequence is 5′ of the target gene and the second nucleic acid sequence is 3′ of the target gene.

In some embodiments, a recombinant nucleic acid molecule disclosed herein comprises: a first nucleic acid sequence of a first regulatory region; and a second nucleic acid sequence of a second regulatory region, wherein said first region comprises a nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 2 Kb upstream but not including the start codon of the UBQ10, and wherein said second region comprises nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 1 Kb downstream from but not including the stop codon region of UBQ10, and wherein said first and second nucleic acid sequences are operably linked to a nucleic acid sequence of a multiple cloning site (MCS), wherein the first nucleic acid sequence is 5′ of the MCS and the second nucleic acid sequence is 3′ of the MCS.

A skilled artisan would appreciate that the term “multiple cloning site” (MCS) may be used interchangeable with the term “polylinker region” having all of the same meanings and qualities. The skilled artisan would appreciate that an MCS encompasses a DNA region within a recombinant nucleic acid molecule that contains multiple unique restriction enzyme cut sites. Recombinant nucleic acid molecules may in some embodiments may be comprised in a plasmid or expression vector. Inclusion of MCS is very useful in biotechnology, allowing for foreign DNA to be inserted into the recombinant nucleic acid, for example but not limited to an expression vector. In some embodiments, the foreign DNA comprises a target gene disclosed herein. This enables the expression vector to act as a vector to insert DNA into another cell, for example but not limited to, for use in targeted gene editing.

In some embodiments, a MCS is suitable for cloning a nucleic acid encoding a target gene within the recombinant nucleic acid molecule. In some embodiments, a MCS comprises a short segment of DNA which contains many (up to ˜20) restriction sites. Restriction sites within an MCS are typically unique to the plasmid or expression vector within which a recombinant nucleic acid molecule would be comprised, occurring only once within the plasmid or expression vector. The nucleic acid sequences of MCS are well known to one of skilled in the art.

In some embodiments, disclosed herein is a recombinant nucleic acid molecule comprising: a first nucleic acid sequence of a first regulatory region: and a second nucleic acid sequence of a second regulatory region, wherein said first region is adjacent to the nucleic acid sequence encoding a UBQ10 extending about 2 Kb upstream but not including the start codon of the UBQ10, and wherein said second region is adjacent to the nucleic acid sequence encoding a UBQ10 extending about 1 Kb downstream from but not including the stop codon region of UBQ10, and wherein said first and second nucleic acid sequences are fused to a nucleic acid sequence of a target gene.

In some embodiments, disclosed herein is a recombinant nucleic acid molecule comprising: a first nucleic acid sequence of a first regulatory region; and a second nucleic acid sequence of a second regulatory region, wherein said first region comprises a nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 2 Kb upstream but not including the start codon of the UBQ10, and wherein said second region comprises nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 1 Kb downstream from but not including the stop codon region of UBQ10, and wherein said first and second nucleic acid sequences are operably linked to a nucleic acid sequence of a target gene.

In some embodiments, a first regulatory region comprises the nucleotide sequence directly 5′ to the ATG start codon of a POLYUNBIQUITIN10 gene starting from the nucleotide 5′ to the ATG. In some embodiments, a first regulatory region comprises a nucleotide sequence upstream of the ATG start codon of a POLYUNBIQUITIN10 gene. In some embodiments, a first regulatory region comprises the about 2 Kb nucleotide sequence upstream of the ATG start codon of a POLYUNBIQUITIN10 gene. In some embodiments, a first regulatory region comprises the about 2 Kb nucleotide sequence directly upstream of the ATG start codon of a POLYUNBIQUITIN10 gene, starting from the nucleotide 5′ to the ATG.

The downstream region of the UBQ10 may include a 3′ untranslated region (UTR), an intron, or a terminator region, or a combination thereof. A skilled artisan would appreciate that the terms “downstream” and “3 prime (3′)” may in some embodiments be used interchangeably herein having all the same qualities and meanings.

In some embodiments, a second nucleic acid sequence of a second regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 27) gttgtggttgtctggttgcgtctgttgcccgttgtctgttgcccattgtg gtggttgtgtttgtatgatggtcgttaaggatcatcaatgtgttttcgct ttttgttccattctgtttctcatttgtgaataataatggtatctttatga atatgcagtttgtggtttcttttctgattgcagttctgagcattttgttt ttgcttccgtttactataccacttacagtttgcactaatttagttgatat gcgagccatctgatgtttgatgattcaaatggcgtttatgtaactcgtac ccgagtggatggagaagagctccattgccggtttgtttcatgggtggcgg agggcaactcctgggaaggaacaaaagaaaaaccgtgatacgagttcatg ggtgagagctccagcttgatcccttctctgtcgatcaaatttgaattttt ggatcacggcaggctcacaagataatccaaagtaaaacataatgaatagt acttctcaatgatcacttatttttagcaaatcagcaattgtgcatgtcaa atgatttcggtgtaagagaaagagttgatgaatcaaaatatctgtagctg gatcaagaatctgaggcagttgtatgtatcaatgatctttccgctacaat gatgttagctatccgagtcaaattgttgtagaattgcatacttcggcatc acattctggatgacataataaataggaagtcttcagatccctaaaaaatt gagagctaataacattagtcctagatgtaactgggtgacaaccaagaaag agacatgcaaatactacttttgtttgaaggagcatccctggtttgacata ttttttctgaatatcaaactttgaaactctacctagtctaatgtctaacg acagatcttactggtttaactgcagtgatatctactatcttttggaatgt tttctccttcagttatacatcaagttccaagatgcaggtgtgcttgattg atgtacatggctgtgagaagtgcatcctgatgttcagatgatggttcatt ctaatgtcttttccttcaatcagttttctcagtctgacttagcttgtttc atctgcatgtttgaatgttcgtttactcatagtaattgcatttttgtagc agaacatatcattggtcatggtttcaactgtgcgcgagtcttatgcttat tcaaactaggaaagcctccgtctagagggtacacgagttgttgctctgtg tgcgtcagtccatagtattaatcttgctagttgtagtatattgtttatgt ggactcggaattcatcatatgctccttctttgcatcaagtaaggcaaggt aatgtatagaagctttttaactctttcatggaagctggcctttgccagca taccatccagaagatatcaaccctgcatcttggctgccg.

In some embodiments, a second nucleic acid sequence of a second regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 28) ttgcagttctgagcattttgtttttgcttccgtttactataccacttaca gtttgcactaatttagttgatatgcgagccatctgatgtttgatgattca aatggcgtttatgtaactcgtacccgagtggatggagaagagctccattg ccggtttgtttcatgggtggcggagggcaactcctgggaaggaacaaaag aaaaaccgtgatacgagttcatgggtgagagctccagcttgatcccttct ctgtcgatcaaatttgaatttttggatcacggcaggctcacaagataatc caaagtaaaacataatgaatagtacttctcaatgatcacttatttttagc aaatcagcaattgtgcatgtcaaatgatttcggtgtaagagaaagagttg atgaatcaaaatatctgtagctggatcaagaatctgaggcagttgtatgt atcaatgatctttccgctacaatgatgttagctatccgagtcaaattgtt gtagaattgcatacttcggcatcacattctggatgacataataaatagga agtcttcagatccctaaaaaattgagagctaataacattagtcctagatg taactgggtgacaaccaagaaagagacatgcaaatactacttttgtttga aggagcatccctggtttgacatattttttctgaatatcaaactttgaaac tctacctagtctaatgtctaacgacagatcttactggtttaactgcagtg atatctactatcttttggaatgttttctccttcagttatacatcaagttc caagatgcaggtgtgcttgattgatgtacatggctgtgagaagtgcatcc tgatgttcagatgatggttcattctaatgtcttttccttcaatcagtttt ctcagtctgacttagcttgtttcatctgcatgtttgaatgttcgtttact catagtaattgcatttttgtagcagaacatatcattggtcatggtttcaa ctgtgcgcgagtcttatgcttattcaaactaggaaagcctccgtctagag ggtacacgagttgttgctctgtgtgcgtcagtccatagtattaatcttgc tagttgtagtatattgtttatgtggactcggaattcatcatatgctcctt ctttgcatcaagtaaggcaaggtaatgtatagaagctttttaactctttc atggaagctggcctttgccagcataccatccagaagatatcaaccctgca tcttggctgccg.

In some embodiments, a second nucleic acid sequence of a second regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 29) gttgtggttgtctggttgcgtctgttgcccgttgtctgttgcccattgtg gtggttgtgtttgtatgatggtcgttaaggatcatcaatgtgttttcgct ttttgttccattctgtttctcatttgtgaataataatggtatctttatga atatgcagtttgtggtttcttttctga.

In some embodiments, a second nucleic acid sequence of a second regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 38) gttgcctgttgttggttgtcgtgttgtctggctgtgtctgttgcccattg tggtggttatgtgtttgcattatggtcttaaaggatcatcaatgtgtttt cgctttctgttcctttctgtttctcatttgtgaataataatggcgtcttt atgaacatccaatttctggtttcttttctgatcgcagtttgagtatttgt ttttgcttttgcctccgtctattacaccactttgcaattactataatata ctaaaagccttcgatccatcttctgtttgatgattcgaatggtatttatt taactcatacccaagtgaagcataaagttagaggagagttcctgttccat tgcctgtttgtatcatgagcaactcatgttaataaacataagaaaaacca tgatgcaatctgtgtagctgatagactttgatgacagacgactcataagt aacaagagataacaaagaggaaacataataaacatgtacgggaagtcctc caacaatgactataatcacatgtttttgtagattagcaattgtacatgtc aaatgatcttggattaaggaaggagcttgtgaatcaaaacatctgaattt ggacctagagtcttgaggtgatcgtactttggatggagagaccatgaata agaataaatgaatctggaactgagaactaaatggaagacacactgatcca acagattaagcttatgacattaatcacagaaggtaactcggtgacaacca agaacggggagctgcaaattctattgtcttaacaacggacctttactggt ttaactgttatgatgtcttttataggtggcttttgggttgttcttcgctc tatccttttatgtaactttcaagaaccaaccaaatgcaggtgttctagat agatatacgtggcatgtgagaagggaccctgaagttcagatgacggt.

In some embodiments, a second nucleic acid sequence of a second regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 39) gttgcctgttgttggttgtcgtgttgtctggctgtgtctgttgcccattg tggtggttatgtgtttgcattatggtcttaaaggatcatcaatgtgtttt cgctttctgttcctttctgtttctcatttgtgaataataatggcgtcttt atgaacatccaatttctggtttctttt.

In some embodiments, a second nucleic acid sequence of a second regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 45) atctcgtctctgttatgcttaagaagttcaatgtttcgtttcatgtaaaa ctttggtggtttgtgttttggggccttgtataatccctgatgaataagtg ttctactatgtttccgttcctgttatctctttctttctaatgacaagtcg aacttcttctttatcatcgcttcgtttttattatctgtgcttcttttgtt taatacgcctgcaaagtgactcgactctgtttagtgcagttctgcgaaac ttgtaaatagtccaattgttggcctctagtaatagatgtagcgaaagtgt tgagctgttgggttctaaggatggcttgaacatgttaatcttttaggttc tgagtatgatgaacattcgttgttgctaagaaatgcctgtaatgtcccac aaatgtagaaaatggttcgtacctttgtccaagcattgatatgtctgatg agaggaaactgcaagatactgagcttggtttaacgaaggagaggcagttt cttccttccaaagcatttcatttgacaatgccttgatcatcttaagtaga gtttctgttgtggaaagtttgaaactttgaagaaacgactctcaagtaaa ttgatgatcacaagtgaaagtgtatgttacataagtggatatttcaccct ttttccatcaatcaaaacatcatatagtaatccattggtttatacaaaca tcaaaatacatttacctctgaaatgaggaaaaaaatgcaaagagattttt gaaaatttccaacaaatg.

In some embodiments, a second nucleic acid sequence of a second regulatory region comprises the following nucleic acid sequence:

(SEQ ID NO: 46) atctcgtctctgttatgcttaagaagttcaatgtttcgtttcatgtaaaa ctttggtggtttgtgttttggggccttgtataatccctgatgaataagtg ttctactatgtttccgttcctgttatctctttctttctaatgacaagtcg aacttcttctttatcatcgcttcgtttttattatctgtgcttcttttgtt taatacgcctgcaaagtgactcgactctgtttagtgcagttctgcgaaac ttgtaaatagtccaattgttggcctctagtaatagatgtagcgaaagtgt tgagctgttgggttctaaggatggcttgaacatgttaatcttttaggttc tgagtatgatgaacattcgttgttgc.

In some embodiments, a second nucleic acid sequence of a second regulatory region comprises a homolog of any one of SEQ ID NOs: 27, 28, 29, 38, 39, 45, or 46. In one embodiment, a second nucleic acid sequence of a second regulatory region is at least 70% homologous to any one of SEQ ID NOs: 27, 28, 29, 38, 39, 45, or 46. In another embodiment, a second nucleic acid sequence of a second regulatory region is at least 80% homologous to any one of SEQ ID NOs: 27, 28, 29, 38, 39, 45, or 46. In another embodiment, a second nucleic acid sequence of a second regulatory region is at least 90% homologous to any one of SEQ ID NOs: 27, 28, 29, 38, 39, 45, or 46. In another embodiment, a second nucleic acid sequence of a second regulatory region is at least 95% homologous to any one of SEQ ID NOs: 27, 28, 29, 38, 39, 45, or 46. In another embodiment, a second nucleic acid sequence of a second regulatory region is at least 98% homologous to any one of SEQ ID NOs: 27, 28, 29, 38, 39, 45, or 46. In another embodiment, a second nucleic acid sequence of a second regulatory region is at least 99% homologous to any one of SEQ ID NOs: 27, 28, 29, 38, 39, 45, or 46.

In some embodiments, a second nucleic acid sequence of a second regulatory region comprises a combination of any one of SEQ ID NOs: 27, 28, 29, 38, 39, 45, or 46.

In some embodiments, a nucleic acid sequence comprising a second nucleic acid sequence of a second regulatory region comprises a contiguous portion of a chromosome. In some embodiments, a nucleic acid sequence comprising a second nucleic acid sequence of a second regulatory region comprises a non-contiguous portion of a chromosome.

In some embodiments, a second regulatory region comprises the nucleotide sequence directly 3′ to the stop codon of a POLYUNBIQUITIN10 gene starting from the nucleotide 3′ to the stop codon. In some embodiments, a second regulatory region comprises a nucleotide sequence downstream of the stop codon of a POLYUNBIQUITIN10 gene. In some embodiments, a second regulatory region comprises the about 1 Kb nucleotide sequence downstream of the stop codon of a POLYUNBIQUITIN10 gene. In some embodiments, a second regulatory region comprises the about 1 Kb nucleotide sequence directly downstream of the stop codon of a POLYUNBIQUITIN10 gene, starting from the nucleotide 3′ to the stop codon.

In some embodiments, a first nucleic acid sequence of a first regulatory region and a second nucleic acid from a second regulatory region comprises upstream and downstream nucleotide sequences of the same POLYUNBIQUITIN10 gene. In some embodiments, a first nucleic acid sequence of a first regulatory region and a second nucleic acid from a second regulatory region comprises upstream and downstream nucleotide sequences of different POLYUNBIQUITIN10 genes.

In some embodiments, a recombinant nucleic acid molecule comprises a first nucleic acid sequence of a first regulatory region and a second nucleic acid sequence from a second regulatory region of the same POLYUNBIQUITIN10 gene, wherein the first and second nucleic acid sequences are operably linked to a nucleic acid sequence of a target gene, wherein the first nucleic acid sequence is 5′ of the target gene and the second nucleic acid sequence is 3′ of the target gene. In some embodiments, a recombinant nucleic acid molecule comprises a first nucleic acid sequence of a first regulatory region and a second nucleic acid from a second regulatory region of different POLYUNBIQUITIN10 genes operably linked to a nucleic acid sequence of a target gene wherein the first nucleic acid sequence is operably linked 5′ of the target gene and the second nucleic acid sequence is operably linked 3′ of the target gene.

In some embodiments, a first nucleic acid sequence of a first regulatory region and a second nucleic acid from a second regulatory region of a POLYUNBIQUITIN10 gene, comprise a promoter-terminator cassette, wherein the first nucleic acid sequence of a first regulatory region comprises a promoter and the second nucleic acid from a second regulatory region comprises a terminator region, and a gene of interest may be inserted between the promoter and terminator regions.

In some embodiments, a recombinant nucleic acid molecule comprises a first nucleic acid sequence of a first regulatory region comprising the nucleic acid sequence of any one of SEQ ID NO: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49, or a homolog thereof, and a second nucleic acid from a second regulatory region comprising the nuclide acid sequence of any one of SEQ ID NO: 27, 28, 29, 38, 39, 45, or 46, or a homolog thereof wherein the first nucleic acid sequence is operably linked 5′ of the target gene and the second nucleic acid sequence is operably linked 3′ of the target gene. In some embodiments, a recombinant nucleic acid molecule comprises a first nucleic acid sequence of a first regulatory region comprising the nucleic acid sequence set forth in SEQ ID NO: 23, or a homolog thereof, and a second nucleic acid from a second regulatory region comprising the nuclide acid sequence set forth in SEQ ID NO: 27, or a homolog thereof, wherein SEQ ID NO: 23 is operably linked 5′ of the target gene and SEQ ID NO: 27 is operably linked 3′ of the target gene.

In some embodiments, a target gene is operably linked to a first nucleic acid sequence of a first regulatory region without any intervening sequences. Examples include but are not limited to, the ATG start codon of a target gene being directly fused to the 3′ nucleotide of any one of SEQ ID NO: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49, or a homolog thereof.

In some embodiments, the ATP start codon of a target gene is directly fused to the 3′ nucleotide of SEQ ID NO: 23.

In some embodiments, a target gene is operably linked to a first nucleic acid sequence of a first regulatory region, wherein intervening nucleotides are present between the ATP start codon of the target gene and the 3′ nucleotide of the first nucleic acid sequence of a first regulatory region. Examples include, but are not limited to, the ATG start codon of a target gene being operably linked to the 3′ nucleotide of any one of SEQ ID NO: 23, 24, 25, 26, 34, 35, 36, 37, 41, 41, 43, 44, 48, or 49, or a homolog thereof, wherein intervening nucleotides are present between the ATP start codon of the target gene and the 3′ nucleotide of the first nucleic acid sequence of a first regulatory region. In some embodiments, the ATP start codon of a target gene is operably linked to the 3′ nucleotide of SEQ ID NO: 23, wherein intervening nucleotides are present between the ATP start codon of the target gene and the 3′ nucleotide of SEQ ID NO: 23.

In some embodiments, a target gene is operably linked to a second nucleic acid sequence of a second regulatory region without any intervening sequences. Examples include but are not limited to, the stop codon of a target gene being directly fused to the 5′ nucleotide of any one of SEQ ID NO: 27, 28, 29, 38, 39, 45, or 46, or a homolog thereof. In some embodiments, the stop codon of a target gene is directly fused to the 5′ nucleotide of SEQ ID NO: 27.

In some embodiments, a target gene is operably linked to a second nucleic acid sequence of a second regulatory region, wherein intervening nucleotides are present between the stop codon of the target gene and the 5′ nucleotide of the second nucleic acid sequence of a second regulatory region. Examples include, but are not limited to, the stop codon of a target gene being operably linked to the 5′ nucleotide of any one of SEQ ID NO: 27, 28, 29, 38, 39, 45, or 46, or a homolog thereof, wherein intervening nucleotides are present between the stop codon of the target gene and the 5′ nucleotide of the second nucleic acid sequence of a second regulatory region. In some embodiments, the stop codon of a target gene is operably linked to the 5′ nucleotide of SEQ ID NO: 27, wherein intervening nucleotides are present between the stop codon of the target gene and the 5′ nucleotide of SEQ ID NO: 27.

In some embodiments, a stop codon comprises a TAA, TAG, or TGA codon.

In some embodiments, a recombinant nucleic acid molecule disclosed herein comprises: a first nucleic acid sequence of a first regulatory region; and a second nucleic acid sequence of a second regulatory region, wherein said first region comprises a nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 2 Kb upstream but not including the start codon of the UBQ10, and wherein said second region comprises nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 1 Kb downstream from but not including the stop codon region of UBQ10, and wherein said first and second nucleic acid sequences are operably linked to a nucleic acid sequence of a target gene, wherein the first nucleic acid sequence is 5′ of the target gene and the second nucleic acid sequence is 3′ of the target gene.

In some embodiments, a recombinant nucleic acid molecule disclosed herein comprises: a first nucleic acid sequence of a first regulatory region; and a second nucleic acid sequence of a second regulatory region, wherein said first region comprises a nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 2 Kb upstream but not including the start codon of the UBQ10, and wherein said second region comprises nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 1 Kb downstream from but not including the stop codon region of UBQ10, and wherein said first and second nucleic acid sequences are operably linked to a nucleic acid sequence of a multiple cloning site (MCS), wherein the first nucleic acid sequence is 5′ of the MSC and the second nucleic acid sequence is 3′ of the MCS.

One of skilled in the art is well aware of the genetic elements that need to be present on the vector in order to successfully transform, select and propagate host cells containing the sequence of interest, for example a target gene. The sequence of interest can be operably linked to one or more promoter sequences a vector.

In some embodiments, an expression vector comprises a recombinant nucleic acid molecule as described herein. In certain embodiments, a recombinant nucleic acid molecule described herein forms part of an expression vector comprising all necessary elements for expression of the target gene. In certain embodiments, a recombinant nucleic acid molecule described herein forms part of an expression vector comprising all necessary elements for expression of a target gene that could be inserted in the MCS. In some embodiments, a library comprises a recombinant nucleic acid molecule as described herein.

Constructs and vectors may also include a transit peptide for targeting of a gene to a plant organelle, for example, to a chloroplast, leucoplast or other plastid organelle. The expression cassettes or the construct disclosed herein may be included in a host cell, plant cell, seed, agricultural product or plant.

In some embodiments, disclosed herein is a recombinant nucleic acid molecule comprising a first nucleotide sequence encoding a gene of interest, wherein said gene of interest is operably linked to a regulatory sequence of the genes encoding chlorophyll a/b binding protein (CAB), chlorophyll a/b binding protein 1 (CAB1), or a combination thereof.

Expression of a Nuclease System Operably Linked to a Ubiquitin Promoter

In some embodiments, a target gene comprises any gene to be expressed. In some embodiments, the target gene encodes a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing or a homolog thereof.

In some embodiments, the target gene encodes a CRISPR associated protein 9 (CAS9) or a homolog thereof. In some embodiments, the target gene is a wild-type CAS9, or a homolog thereof. In some embodiments, the target gene is a mutant CAS9 or a homolog thereof. Numerous mutations modulating CAS9 function are known in the art. In a one exemplary embodiment, the target gene is CAS9 comprising a mutation in RuvC domain, for example CAS9-D10A. In another exemplary embodiment, the target gene is CAS9 comprising a mutation in HNH domain, for example CAS9-H840A. The nucleic acid and amino acid sequences of wild-type CAS9 and of numerous CAS9 mutants are well known in the art and publicly available in genetic sequence databases.

In some embodiments, a target gene described herein comprise homologs, analogs, or orthologs thereof. In some embodiments, target genes comprise functional fragments, functional variants, or functional derivatives encoding functional fragments, functional variants, or functional derivatives of a nickase, or a CRISPR-associated endonuclease (CAS nuclease), or a DNA endonuclease enzyme used in targeted gene editing.

In some embodiments, target genes are 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequences of the genes known in the art. In one embodiment, a target gene comprises sequences that are complementary to the sequences of the genes encoding a nickase, or a CRISPR-associated endonuclease (CAS nuclease), or a DNA endonuclease enzyme used in targeted gene editing known in the art. In another embodiment, target genes comprise sequences of the genes encoding a nickase, or a CRISPR-associated endonuclease (CAS nuclease). or a DNA endonuclease enzyme used in targeted gene editing that hybridize, for example, under stringent conditions, to the sequences of the genes known in the art.

In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing by about 15-1000 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence. In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing by about 15-750 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence. In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing by about 15-500 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence.

In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing by about 15-250 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence. In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease. or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing by about 50-200 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence.

In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing by about 100-250 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence. In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing by about 100-400 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence.

In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing by about 250-500 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence.

In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing by about 250-750 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence. In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing by about 100 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence.

In some embodiments, use of a recombinant nucleic acid molecule comprising a UBQ10 regulatory sequence, increases the content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing used in targeted gene editing in at least 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 fold compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence.

In some embodiments, use of a recombinant nucleic acid molecule disclosed herein, increases target mutagenesis by 100% compared with a corresponding nucleic acid molecule not comprising an UBQ10 regulatory sequence.

In some embodiments, a target gene comprises a guiding RNA (gRNA). In some embodiments a target gene comprises a gRNA and a nuclease.

Recombinant Nucleic Acid Molecules

Specifically, disclosed herein is a recombinant nucleic acid molecule comprising a first nucleotide sequence encoding a target gene, wherein said target gene is operably linked to a UBQ10 regulatory sequence. In some embodiments, said target gene comprises a nuclease system gene, or a part thereof.

In some embodiments, disclosed herein is a recombinant nucleic acid molecule comprising a first nucleotide sequence encoding a nuclease system, wherein said nuclease system is targeted to a plant endogenous DNA sequence, and wherein at least one component of said nuclease system is operably linked to a UBQ10 regulatory sequence. In some embodiments, the nucleic acid molecule further comprises a second nucleotide sequence encoding a viral replicon comprising a donor nucleic acid sequence targeted to a plant endogenous DNA sequence.

Some of the embodiments of the replicon and its components are detailed above in “Methods of Gene targeting Section” and are incorporated herein by reference. In some embodiments, a replication initiator protein (Rep) of the replicon is operably linked to a promoter. In some embodiments, said promoter is a constitutive promoter. In some embodiments, said promoter is a tissue-specific promoter, which restricts Rep expression to any tissue of interest. In some embodiments, said promoter comprises a CAMV 35S promoter.

In some embodiments, a donor nucleic acid sequence is located between the LIRs. In some embodiments, a nucleic acid encoding a nuclease system is between the LIRs. In some embodiments, a nucleic acid encoding a guiding RNA (gRNA) is between the LIRs. In some embodiments, a donor nucleic acid sequence, a nucleic acid encoding a nuclease system, and a nucleic acid encoding a gRNA are located between the LIRs. In some embodiments, a donor nucleic acid sequence and a nucleic acid encoding a nuclease system are located between the LIRs. In some embodiments, a donor nucleic acid sequence and a nucleic acid encoding a gRNA are located between the LIRs. In some embodiments, a nucleic acid encoding a nuclease system and a nucleic acid encoding a gRNA are located between the LIRs.

In some embodiments, a donor nucleic acid sequence is operably linked to a UBQ10 regulatory sequence. In some embodiments, a nucleic acid encoding a nuclease system is operably linked to a UBQ10 regulatory sequence. In some embodiments, a nucleic acid encoding a gRNA is operably linked to a UBQ10 regulatory sequence.

In some embodiments, a replicon, a donor nucleic acid sequence, a nucleic acid encoding a nuclease system, and a nucleic acid encoding a gRNA are all located in a same recombinant nucleic acid molecule or expression vector. A skilled artisan would appreciate, however, that all these elements should not be necessarily located in a same nucleic acid molecule or expression vector. In some embodiments, a donor nucleic acid sequence is located in a second nucleic acid molecule or expression vector. In some embodiments, a nucleic acid encoding a nuclease system is located in a second nucleic acid molecule or expression vector. In some embodiments, a nucleic acid encoding a gRNA is located in a second recombinant nucleic acid molecule or expression vector. In some embodiments, these elements are located in 3 separate recombinant nucleic acid molecules or expression vector.

In some embodiments, a nucleic acid encoding a selection marker is further included in the expression vector. A number of selection markers are known in the literature, and a skilled artisan would appreciate how to incorporate them in the vectors described here. In some embodiments, a selection marker comprises neomycin phosphotransferase II (NPTII), which confers resistance to kanamycin, neomycin, geneticin (G418), and paromomycin.

A skilled artisan would appreciate that the terms “nucleic acid sequence” and “nucleotide sequence” may be used interchangeably having all the same qualities and meanings. A skilled artisan would appreciate that the terms “expression vector”, “expression construct”, “recombinant nucleic acid” are, in some embodiments, used interchangeably having all the same qualities and meanings. In one embodiment, a “genomic polynucleotide sequence” refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

In one embodiment, a “composite polynucleotide sequence” refers to a sequence, which is comprised of difference nucleotide sequences that represent non-contiguous portions of a chromosome. In one embodiment, a composite sequence can include nucleic acid sequences from multiple sources. In one embodiment, a composite sequence can include nucleic acid sequences from multiple genes. In one embodiment, a composite sequence can include nucleic acid sequences from a single gene, wherein the sequence does not represent a contiguous portion of a chromosome.

In some embodiments, the methods for gene targeting disclosed herein comprise the use of any of the recombinant nucleic acid molecules described in this section.

Genetically Modified Plants

In some embodiments, a genetically modified plant described herein comprises at least one cell comprising a recombinant nucleic acid molecule as disclosed herein.

A genetically modified plant comprising at least one cell comprising a recombinant nucleic acid molecule as disclosed herein, may in some embodiments first be selected based on the expression of the target gene or protein encoded by the target gene. In some embodiments, a genetically modified plant comprising at least one cell comprising a recombinant nucleic acid molecule disclosed herein, may in some embodiments first be selected based on an increased content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing or a homolog thereof In some embodiments, a genetically modified plant comprising at least one cell comprising a recombinant nucleic acid molecule disclosed herein, may in some embodiments first be selected based on an increased mutagenesis activity compared with an unmodified plant. In some embodiments, a genetically modified plant comprising at least one cell comprising a recombinant nucleic acid molecule disclosed herein, may in some embodiments first be selected based on an increased gene targeting activity compared with an unmodified plant.

In some embodiments, a genetically modified plant is a Solanaceous crop plant. In some embodiments, a Solanaceae crop plant is selected from the group comprising a cultivated tomato plant, a wild-tomato plant, a cultivated potato plant, a wild-potato plant, an aubergine plant, a chili pepper plant, and a bell pepper plant. In some embodiments, the expression of a target gene is increased compared to its expression in a corresponding unmodified plant.

In some embodiments, an expression vector is operably linked to an additional promoter sequence so that the expression of the targeting molecule can be controlled under different conditions. In another embodiment, the targeting gene is operably linked to a constitutive promoter. In another embodiment, the targeting gene is operably linked to an inducible promoter. In another embodiment, the targeting gene is operably linked to a tissue active or specific promoter. In another embodiment, the targeting gene is operably linked to a developmental-stage active or specific promoter. When the targeting gene is linked to a constitutive promoter, changes in expression of a gene will be observed in all tissues and at all times and a broad overview of the effects of the expression of the gene on a plant will be observed. When the targeting gene is linked to a tissue specific promoter or an inducible promoter or developmental-stage promoter, the expression of the targeting gene may be turned on or off in a particular tissue such as seed, roots, flowers, leaves, shoots, fruits or stems, during a particular period in development, such as early, middle or late stages in development, or under particular conditions, such as specific environmental or disease stresses.

A skilled artisan would appreciate that early stages of plant development may encompass the time before flowering, which would encompass the time after moving from the tissue culture to soil (in a laboratory situation). A skilled artisan would appreciate that late stage, also referred to as “an older stage” or “older plant”, of plant development may encompass a time after early stage. Plant development may also be described wherein a first stage may encompass seed germination, a second stage may encompass a vegetative growth stage, a third stage may encompass a reproductive stage wherein flowers and fruit are produced.

In some embodiments, a plant may be transformed with more than one expression vector. In some embodiments, an additional expression vector comprises nucleic acid sequence encoding a guide RNA molecule. In some embodiments, an additional expression vector comprises nucleic acid sequence encoding other elements of a targeted gene editing system.

In some embodiments, the genetically modified plant comprises increased gene targeting activity compared to a corresponding unmodified plant.

In some embodiments, a cell comprises a recombinant nucleic acid molecule as described herein. In some embodiments, the cell is a plant cell. In some embodiments, the plant cell is a Solanaceae plant cell. In some embodiments, the plant cell is a Solanaceae crop plant cell. In some embodiments, a Solanaceae crop plant comprises cultivated tomato plant, a wild-tomato plant, a cultivated potato plant, a wild-potato plant, an aubergine plant, a chili pepper plant, and a bell pepper plant. In some embodiments, the cell is an algae cell.

Methods for Generating a Genetically Modified Plant

In some embodiments, disclosed herein is a method for producing a transgenic plant seed, the method comprising: (a) introducing into at least one cell of a plant a first nucleic acid comprising a viral replicon comprising a donor nucleic acid sequence, said donor sequence targeted to a plant endogenous DNA sequence; and (b) introducing into the cell of (a) a nucleic acid comprising a nuclease system, wherein said nuclease system is targeted to said plant endogenous DNA sequence, and wherein at least one component of said nuclease system is expressed under a UBQ10 regulatory sequence; (c) generating a transgenic plant from said at least one cell; and (d) growing said transgenic plant to obtain a seed.

In some embodiments, methods for generating a transgenic plant or alga, comprise transforming a plant or algae cell with a nucleic acid molecule disclosed herein or an expression vector comprising a nucleic acid molecule disclosed herein. The transfer of foreign genes into the genome of a plant or algae is called transformation. Methods for transforming a plant cell with nucleic acids sequences are well known in the art, as are methods of transforming algae.

Transformation of plant or algae species is now a fairly routine technique. As used herein the term “transformation” or “transforming” may encompass in some embodiments, a process by which a foreign DNA, such as a recombinant nucleic acid molecule disclosed herein, or an expression vector comprising a recombinant nucleic acid molecule disclosed herein, enters and changes a recipient cell into a transformed, genetically modified or transgenic cell. Transformation may be stable, wherein the nucleic acid sequence is integrated into the plant genome and as such represents a stable and inherited trait, or transient, wherein the nucleic acid sequence is expressed by the cell transformed but is not integrated into the genome, and as such represents a transient trait. In some embodiments, the recombinant nucleic acid molecule disclosed herein is stably transformed into a plant cell.

A transgenic plant is then grown under conditions suitable for the expression of the target gene. The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (See Weissbach and Weissbach, In.: Methods for Plant Molecular Biology, (Eds.), 1988 Academic Press, Inc., San Diego, Calif.). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

Markers or other techniques, known to one of skilled in the art, can be used to determine whether the transgenic molecule is stably integrated into the genome of said plant.

The regenerated plants containing the foreign, exogenous gene that encodes a target gene or a homolog thereof, can then be further propagated as is well known in the art. The particular method of propagation will depend on the starting plant tissue and the particular plant species to be propagated.

In some embodiments, the generated transformed plants are clonally propagated. In some embodiments, the generated transformed plants are propagated by classical breeding techniques. In some embodiments, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines, or pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant containing a desired target gene may then be cultivated using methods well known to one of skill in the art.

In some embodiments, transgenic plants can be observed or tested for whether the transgenic molecule is effective in inducing floral fate or its associated phenotypic trait in the transgenic plants.

Throughout this application a plant, plant part, seed or plant cell transformed with, or transformed by, a construct or a nucleic acid is to be understood as meaning a plant, plant part, seed or plant cell that carries said construct or said nucleic acid as a transgene due the result of an introduction of said construct or said nucleic acid by biotechnological means. The plant, plant part, seed or plant cell therefore comprises said recombinant construct or said recombinant nucleic acid. Any plant, plant part, seed or plant cell that no longer contains said recombinant construct or said recombinant nucleic acid after introduction in the past, is termed null-segregant, nullizygote or null control, but is not considered a plant, plant part, seed or plant cell transformed with said construct or with said nucleic acid within the meaning of this application.

In some embodiments, methods disclosed herein are for producing a transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of a stably-integrated recombinant DNA construct. In some embodiments, an enhanced trait is selected from the group comprising increased expression of a target gene; increased content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing or a homolog thereof: increased mutagenesis: increased gene targeting: or any combination thereof.

In some embodiments, provided herein is a method for generating a transgenic plant, the method comprising: transforming a cell of the plant substantially the same genetic background with at least one nucleic acid molecule to obtain a recombinant plant cell: and generating a transgenic plant from said recombinant plant cell, wherein said nucleic acid molecule comprises: (a) donor nucleic acid sequence, said donor sequence targeted to a plant endogenous DNA sequence; and/or (b) a nuclease system, wherein said nuclease system is targeted to said plant endogenous DNA sequence, and wherein at least one component of said nuclease system is expressed under a UBQ10 regulatory sequence.

In some embodiment, the method further comprises steps of determining whether the transgenic molecule is stably integrated into the genome of said plant; and determining whether the transgenic molecule is effective in gene targeting in said transgenic plant. In some embodiments, provided herein is a method for producing a seed of a plant, the method comprising: transforming a cell of the plant of substantially the same genetic background with a nucleic acid molecule to obtain a recombinant plant cell; generating a transgenic plant from said recombinant plant cell: and growing said plant to obtain a seed, thereby producing said seed of said plant, wherein said nucleic acid molecule is the recombinant molecule disclosed herein.

In some embodiments, provided herein is a method for generating a transgenic plant, the method comprising: transforming a cell of the plant with a nucleic acid molecule to obtain a recombinant plant cell; and generating a transgenic plant from said recombinant plant cell, wherein said nucleic acid molecule comprises: (a) donor nucleic acid sequence, said donor sequence targeted to a plant endogenous DNA sequence: and (b) a nuclease system targeted to said plant endogenous DNA sequence, and wherein at least one component of said nuclease system is expressed under a UBQ10 regulatory sequence.

In some embodiment, the method further comprises steps of determining whether the transgenic molecules are stably integrated into the genome of said plant: and determining whether the transgenic molecules are effective in gene targeting in said transgenic plant. In some embodiments, provided herein is a method for producing a seed of a plant, the method comprising: transforming a cell of the plant of not substantially the same genetic background with a nucleic acid molecule to obtain a recombinant plant cell: generating a transgenic plant from said recombinant plant cell: and growing said plant to obtain a seed, thereby producing said seed of said plant, wherein said nucleic acid molecule is the recombinant molecule disclosed herein.

In some embodiments, provided herein is a transgenic plant comprising recombinant nucleic acid molecule comprising: a first nucleic acid sequence of a first regulatory region: and a second nucleic acid sequence of a second regulatory region, wherein said first region comprises a nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 2 Kb upstream but not including the start codon of the UBQ10, and wherein said second region comprises nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 1 Kb downstream from but not including the stop codon region of UBQ10, and wherein said first and second nucleic acid sequences are operably linked to a nucleic acid sequence of a target gene, wherein the first nucleic acid sequence is 5′ of the target gene and the second nucleic acid sequence is 3′ of the target gene.

In some embodiments, said target gene comprises: (a) donor nucleic acid sequence, said donor sequence targeted to a plant endogenous DNA sequence: and (b) a nuclease system targeted to said plant endogenous DNA sequence, and wherein at least one component of said nuclease system is expressed under a UBQ10 regulatory sequence.

In some embodiments, provided herein is a transgenic plant comprising recombinant nucleic acid molecule comprising: a first nucleic acid sequence of a first regulatory region; and a second nucleic acid sequence of a second regulatory region, wherein said first region comprises a nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 2 Kb upstream but not including the start codon of the UBQ10, and wherein said second region comprises nucleic acid sequence adjacent to the nucleic acid sequence encoding a UBQ10 extending about 1 Kb downstream from but not including the stop codon region of UBQ10, and wherein said first and second nucleic acid sequences are operably linked to a target gene and the second nucleic acid sequence is 3′ of said target gene.

The recombinant nucleic acid molecules described herein are not limited to manipulating coding regions. In some embodiments, the recombinant nucleic acid molecules can be used in modifying promoters for activation or repression of genes.

The term “plant” as used herein may relate to any member of Plantae kingdom, including flowering plants, conifers, ferns, clubmosses, hornworts, liverworts, mosses and the green algae. In some embodiments, the plant is a monocot or dicot plant. Examples of monocot plant includes, but are not limited to, corn, wheat, rice, sugar cane, and banana. Examples of dicot plant includes, but are not limited to, soybean, beans, peas, lentils, peanuts, tomatoes, potatoes, cotton, and perennial fruit trees including grapes, apple, and orange.

In some embodiments, methods described are used to increase gene targeting in a plant, wherein the method comprises transforming at least one plant cell of the plant with the recombinant nucleic acid molecule disclosed herein or an expression vector comprising the recombinant nucleic acid molecule, thereby producing a plant with increasing gene targeting, compared to a corresponding non-transformed plant.

In some embodiments, the method for generating a transgenic plant comprises transforming at least one plant cell of the plant with the recombinant nucleic acid molecule disclosed herein or an expression vector comprising the recombinant nucleic acid molecule, thereby producing a plant with increasing gene targeting, compared to a corresponding non-transformed plant.

In some embodiments, disclosed herein is a method for producing a transgenic seed of a plant, the method comprising: transforming at least one cell of the plant with a recombinant molecule described herein, and generating a transgenic plant from said at least one transformed plant cell: and growing said transgenic plant to obtain a seed, thereby producing said seed of said plant, wherein any plant produced from said seed has at least a mutated gene.

In some embodiments, said mutated gene comprises a donor nucleic acid sequence, said donor sequence targeted to a plant endogenous DNA sequence. In some embodiments, said donor nucleic acid sequence is integrated to the plant genome in a region nicked by a nuclease system targeted to said plant endogenous DNA sequence, wherein at least one component of said nuclease system is expressed under a regulatory sequence. In some embodiments, said regulatory sequence is a UBQ10 regulatory sequence.

In some embodiments, a genetically modified algae described herein comprises at least one cell comprising a recombinant nucleic acid molecule as disclosed herein.

There are various methods of introducing foreign genes into algae (See for example, Radakovits et al., Eukaryotic Cell April 2010 vol. 9(4): 486-501; Radakovits et al., Nat Commun. 2012 Feb. 21; 3: 686; Killan et al., PNAS 2011 vol. 108(52):21265-21269: and Newell, Transgenic Plant Journal. April 2007 vol. 1(1):81-98). Algal transformation methods are fully described in U.S. Pat. Nos. 5,661,017 and 7,001,772, and Application Publication US 2008/0194029.

A genetically modified alga comprising at least one cell comprising a recombinant nucleic acid molecule as disclosed herein, may in some embodiments first be selected based on the expression of the target gene or protein encoded by the target gene. In some embodiments, a genetically modified alga comprising at least one cell comprising a recombinant nucleic acid molecule disclosed herein, may in some embodiments first be selected based on an increased content of a nuclease, a nickase, a ZFN system, a TALEN system, a meganuclease, or a CRISPR/Cas nuclease used in targeted gene editing or a homolog thereof In some embodiments, a genetically modified alga comprising at least one cell comprising a recombinant nucleic acid molecule disclosed herein, may in some embodiments first be selected based on an increased mutagenesis activity compared with an unmodified alga. In some embodiments, a genetically modified alga comprising at least one cell comprising a recombinant nucleic acid molecule disclosed herein, may in some embodiments first be selected based on an increased gene targeting activity compared with an unmodified alga.

In some embodiments, an alga may be transformed with more than one expression vector. In some embodiments, an additional expression vector comprises nucleic acid sequence encoding a guide RNA molecule. In some embodiments, an additional expression vector comprises nucleic acid sequence encoding other elements of a targeted gene editing system.

In some embodiments, the genetically modified alga comprises increased gene targeting activity compared to a corresponding unmodified alga.

The following examples are presented in order to more fully illustrate embodiments disclosed herein. They should in no way be construed, however, as limiting the broad scope disclosed herein.

EXAMPLES Example 1 Identification of Upstream Region to Promote Gene Expression

Objective: As a first step towards optimizing expression of a gene in plants, for example but not limited to the CAS9 gene, the best upstream regulatory region to promote gene expression was sought.

Methods: In order to identify the best upstream region to promote gene expression, initial studies looked for the genes that display highest expression in all plant tissues. To this end, the tomato transcriptome was analyzed and several candidate genes were identified.

Transient expression assays: The region located ˜2 kb upstream the candidate genes was then amplified and fused to the RED FLUORESCENT PROTEIN (RFP) gene, [˜2 kb upstream region-RFP-˜1 kb downstream region]. The reporter RFP gene was then introduced into agrobacterium, and the resulting transformed bacteria were used to inoculate leaves of Nicotiana benthamiana (N. benthamiana). RFP fluorescence was then measured 7 days post inoculation.

The six candidate genes were CHLOROPHYLL A-B BINDING PROTEIN (Solyc03005760; SICABI; SEQ ID NO: 7), CHLOROPHYLL A-B BINDING PROTEIN (Solyc02g071010: SICAB: SEQ ID NO: 10), GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (Solyc04g009030; SIGAPDH; SEQ ID NO: 13), TYPE 2 METALLOTHIONEIN (Solyc09g010800; SIMETALL: SEQ ID NO: 16); POLYUBIQUITIN10 (Solyc07g064130; SlUBIQ10: SEQ ID NO: 22), and RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL CHAIN (Solyc03g034220: SIRBCS; SEQ ID NO: 30).

The plasmids harboring the RFP gene included ˜2 Kb upstream and ˜1 Kb downstream regions from the candidate genes. The upstream and downstream regions for each plasmid used are provided in Table 1:

TABLE 1 Upstream (Promoter) and downstream (Terminator) sequences used in the RFP plasmids Upstream Gene Source & Downstream Gene Source & Name Sequence Sequence SlCAB1 SlCAB1 SEQ ID NO: 8 SlTHI4 SEQ ID NO: 21 SlCAB SlCAB SEQ ID NO: 11 SlTHI4 SEQ ID NO: 21 SlGAPDH SlGAPDH SEQ ID NO: 14 SlTHI4 SEQ ID NO: 21 SlMETAL SlMETAL SEQ ID NO: 17 SlTHI4 SEQ ID NO: 21 SlUBIQ10 SlUBIQ10 SEQ ID NO: 23 SlUBIQ10 SEQ ID NO: 27 SlRBCS SlRBCS SEQ ID NO: 31 SlTHI4 SEQ ID NO: 21

The cauliflower mosaic virus (CaMV) promoter was used as a control

Plasmids harboring the RFP gene and including ˜2 Kb upstream and ˜1 Kb downstream regions from the UBQ10s from other plant species were also constructed. The other plant genes included potato (StUBIQ10, Sotub07g026130, SEQ ID NO: 33), and Arabidopsis thaliana (AtUBIQ10long, AT4G05320, SEQ ID NO: 40: and AtUBIQ10short, AT4G05320, SEQ ID NO: 47). The upstream and downstream regions for each plasmid from the UBQ10s from other plant species are provided in Table 2.

TABLE 2 Upstream (Promoter) and downstream (Terminator) sequences used in the RFP plasmids Upstream Gene Source & Downstream Gene Name Sequence Source & Sequence StUBIQ10 StUBIQ10 SEQ ID NO: 34 StUBIQ10 SEQ ID NO: 38 AtUBIQ10 AtUBIQ10 SEQ ID NO: 41 AtUBIQ10 SEQ ID long long long NO: 45 AtUBIQ10 AtUBIQ10 SEQ ID NO: 48 AtUBIQ10 SEQ ID short short short NO: 45

Results:

It was observed that the region ˜2 kb upstream and ˜1 kb downstream of the SlUBQ10 directed RFP expression in the strongest manner (FIG. 1).

Next, it was determined whether the genomic regions adjacent to the POLYUBIQUITTIN10 of other plant species were also able to direct gene expression in the strongest manner. To this end, the RFP gene was fused to the ˜2 kb upstream and ˜1 kb downstream genomic regions of the UBQ10 of potato (Solanum tuberosum) and Arabidopsis (Arabidopsis thaliana). A short (˜700 bp: SEQ ID NO: 48) and a long (˜1700 bp; SEQ ID NO: 41) genomic region upstream of the Arabidopsis UBQ10 were compared. The capacity of these four genomic regions (SlUBIQ10, StUBIQ10, AtUBIQ10 long. AtUBIQ10short) to direct RFP expression was determined in a transient expression assay on N. benthamiana leaves.

It was observed that in all three species, the regions ˜2 kb upstream and ˜1 kb downstream of the UBQ10 directed RFP expression in the strongest manner (FIG. 2). In addition, RFP expression under the shorter upstream region (SEQ ID NO: 48) of the AtUBQ10 was lower than that of the other genomic regions measured here (FIG. 2).

Conclusion:

The regions ˜2 kb upstream and ˜1 kb downstream of the UBQ10 were found to enhance the expression of sequences adjacent to them. In order to achieve the highest expression of a target gene, for example but not limited to the CAS9 gene, the target can be fused between the regions ˜2 kb upstream and ˜1 kb downstream of the UBQ10.

Example 2 Use of the Adjacent Regions of the UBQ10 for High-Efficiency Expression of a Pepper Gene in Tomato Plants

Objective: To verify that the regions ˜2 kb upstream and ˜1 kb downstream of the UBQ10 induce high expression of a gene fuse amid them in tomato plants. To provide a plant with stable and high expression levels of the VANILIN SYNTHASE gene of Capsicum annum (CaVAN).

Methods:

The VANILIN SYNTHASE gene of Capsicum annum (CaVAN) was fused to the genomic regions located ˜2 kb upstream (SEQ ID NO: 23) and 1 kb downstream (SEQ ID NO: 27) the SlUBQ10 of tomato: [˜2 kb upstream region-CaVAN-˜1 kb downstream region]. and this cassette was introduced into tomato plants. This expression cassette was generated using standard molecular biology procedures, and the tomatoes (Solanum lycopersicum cv. microtom) were transformed using Agrobacterium tumefaciens, as described in the literature. The CaVAN expression was measured by qPCR in transgenic tomato lines (T1).

Results:

The SlUBIQ10 cassette efficiently enhanced the expression of CaVAN. FIG. 3 shows the relative expression levels of the VANILIN SYNTHASE gene of Capsicum annum (CaVAN) observed in 4 transgenic tomato plants containing a cassette consisting of the SlUBIQ10 promoter/terminator fused to the CaVAN coding sequence is presented in.

Conclusion:

The SlUBIQ10 promoter/terminator cassette efficiently enhanced the expression of a non-tomato gene fused into it in tomato plants.

Example 3 Use of the Adjacent Regions of the UBQ10 for High-Efficiency CRISPR-CAS Expression in Tomato Plants

The results presented in Example 1 show that the genomic region located ˜2 kb upstream and (but not necessarily) ˜1 kb downstream the UBQ10, is able to enhance the expression of genes fused into it.

It is desirable obtaining high and stable CAS9 expression in plants. Since plants having high CAS9 expression levels can be efficiently mutagenized. Therefore, in order to obtain high and stable CAS9 expression, the CAS9 gene was fused among the ˜2 kb upstream and ˜1 kb downstream of the UBQ10 gene.

Objective:

To provide a plant with stable and high expression levels of the CAS9 gene, thereby providing means and methods to efficiently mutagenize plants and algae.

Methods:

The genomic region located ˜2 kb upstream the UBQ10 contains a promoter region and the 5′ UTR. Notably, this 5′ UTR is conserved in plants. It is ˜400 to 600 bp long and contains a ˜300-500 bp long intron whose 3′ end is adjacent to the start codon of this gene. The presence of this intron allows high gene expression in eukaryotes, especially when expressing prokaryotic genes, as exon-exon junctions are known to enhance gene expression in eukaryotes. Used herein, the tomato promoter region includes at least SEQ ID NO: 24, and the 5′ UTR including the intron comprises SEQ ID NO: 25 and SEQ ID NO: 26.

To obtain the highest CAS9 expression, the CAS9 gene was fused to the genomic region located ˜2 kb upstream (SEQ ID NO: 23) and 1 kb downstream (SEQ ID NO: 27) the SlUBQ10 of tomato [˜2 kb upstream region-CAS9-˜1 kb downstream region], and this CAS9 cassette was introduced into tomato plants cv. M82 and cv. microtom.

Moreover, in order to define the most suitable expression cassette to direct CAS9 expression, and to obtain the highest mutation rate, the CRISPR-CAS system was used to mutagenize the CRTISO gene (Solyc10g081650, SEQ ID NO: 74). This gene is involved in carotenoid biosynthesis and a homozygous or bi-allelic mutation(s) in this gene causes the plants to display an orange color in the young leaves, in the flowers and in the fruit tissues. Furthermore, in an attempt to improve the mutation rate, the BeYDV Replicon was also used, which generate high levels of CAS9 and gRNA copies (Bakes N J et al. (2014) Plant Cell, 26(1): 151-63).

Early stage results are those results collected after moving the plant from tissue culture to soil and before the flowering of the plant. Late stage results are those results encompassing reproductive growth.

DNA sequence analysis was performed using Illumina sequencing HiSeq 2500 system, high output run, paired-end 125 bp at the G-INCPM.

Results:

The expression level of CAS9 was then determined in the transgenic tomato plants and found to be high (FIGS. 4A and 4B). In this experiment, the mutation rate obtained when the CAS9 gene was overexpressed using the SlPOLYUBIQUITIN10 cassette (SlUBIQ10 promoter region (SEQ ID NO: 23)/SlUBIQ10 terminator region (SEQ ID NO: 27) was compared with the mutation rate when the CAS9 gene was expressed in a Petroselinum crispum UBIQUITIN4 (PcUBIQUITIN4) cassette (See, Fauser, F. et al., The Plant Journal (2014) 79, 348-359).

Several transgenic tomato plants (cv. microtom) were produced that harbored a CRISPR-CAS construct designed to target either the CRTISO gene (Solyc10g081650) or the PSY1 gene (Solyc03g031860). In this experiment, the CAS9 gene was overexpressed either under the control of the PcUBIQUITIN4 cassette, or under the control of the SlPOLYUBIQUITIN10 cassette (SlUBIQ10 promoter region (SEQ ID NO: 23)/SlUBIQ10 terminator region (SEQ ID NO: 27)), with or without the use of the BeYDV Replicon. Then the phenotype (orange color of the flowers for crtiso, and yellow color of the fruits of psy1) of the plants obtained was monitored as an indication of the indel frequency (Table 3).

TABLE 3 Phenotypic analysis of gene targeting of the CRTISO and PSY1 loci in Micro-Tom Wild Type plants T₀ plants T₀ fruit phenotype that gave Red Orange/ Mutated Construct fruit (WT) Yellow Chimera phenotype/total Rep-crtiso- 22 0 22 0  100% ubi4 Rep-psy1- 32 4 7 21 54.7% ubi4 Rep-crtiso- 2 0 2 0  100% ubi10 Rep-psy1- 4 0 2 2   50% ubi10 crtiso-ubi4 28 0 26 2 96.4% psy1-ubi4 32 7 21 4 71.9% crtiso-ubi10 29 1 28 0 96.6% psy1-ubi10 12 4 6 2 58.3%

High frequencies of mutants were observed in the first generation of transformants, and all samples (of all different constructs) were sent for DNAseq analysis to determine indel frequencies (FIGS. 5A and 5B).

Conclusion:

It was determined that the region ˜2 kb upstream and ˜1 kb downstream of the UBQ10 provides a suitable expression cassette to express foreign genes at high levels in plants. In particular, this cassette was very effective fir expressing the CAS9 gene for generating highly efficient gene targeting. Use of the UBQ10 cassette to express the CAS9 gene at an early stage of plant development would be advantageous for the breeder who wishes to mutagenize a given gene in a plant, as it allows the breeder to obtain the desired mutation throughout the plant and in the seeds.

Example 4 General Methods Used in Examples 5-8

Cloning T-DNA Cassettes and Agrobacterium-Mediated Transformation

All T-DNA constructs were built using the Golden Braid (GB) cloning system on pCAMBIA binary vectors (Sarrion-Perdigones et al., Methods Mol. Biol. (2014) 1116, 133-151). All geminiviral components on pLSL and pREP plasmids (Baltes et al., Plant Cell (2014) 26, 151-163) and the CRISPR components, Ubiquitin4-2 promoter from Petroselinum crispum (PcUbi4-2) on pDE-Cas9 plasmid (Fauser et al., Plant J. (2014) 79, 348-359) U6-26:gRNA, synthesized by IDT (Integrated DNA Technologies) and Ubiquitin10 from Solanum lycopersicum (Solyc07g064130) promoter region (2079 bp upstream to the ATG translation initiation codon (SEQ ID NO: 23)) and terminator region (1443 bp downstream to the TAA stop codon (SEQ ID NO: 27)) were cloned to GB vectors. Sequences of the gRNAs were cloned by BbsI digestion and ligation of primers. The primers used for gRNA ligation are shown in Table 4.

TABLE 4 Primers used for gRNA ligation SEQ ID Name Sequence NO: crtiso_t3_Fw 5′-ATTGCGATGCTACCAGCATTCTG 50 crtiso_t3_Rv 5′-GCTACGATGGTCCTAAGACCAAA 51 psy1_t2_Fw 5′-ATTGAGCGTATATAATGCTGCTT 52 psy1_t2_Rv 5′-TCGCATATATTACGACGAACAAA 53 Tmic_t1_Fw 5′-ATTGTTTGATGTTGGTTCATCAGT 54 Tmic_t1_Rv 5′-AAACTACAACCAAGTAGTCACAAA 55 Tmic_t2_Fw 5′-ATTGGTTCATCAGTAGGCCAACT 56 Tmic_t2_Rv 5′-CAAGTAGTCATCCGGTTGACAAA 57

Cotyledon transformation was done using Agrobacterium strain GV3101 as described in Meissner et al. Plant J. (1997), 12, 1465-1472, with minor changes (no tobacco feeder cells layer and with 1 mg zeatin medium for first selection plates, 0.5 mg zeatin, and the addition of 0.5 mg zeatin-riboside medium for the second selection plates and 0.2 mg zeatin for the third selection plates).

Plant Material and Growth Conditions

The gene targeting cassettes, comprising a CRISPR/Cas9 system, a gRNA, a geminiviral replicon, and a kanamycin resistance gene (FIG. 6), were transformed into wild-type (WT) Micro-Tom tomato (Lycopersicon esculentum). Gene targeting cassettes, comprising a CRISPR/Cas9 system, a gRNA, a geminiviral replicon, a donor sequence, and a kanamycin resistance gene (FIG. 7), were transformed into tangerine, a Micro-Tom fast neutron mutant with a 281 bp deletion in the CRTISO gene (Isaacson et al. Plant Cell (2002) 14, 333-42). Control targeted mutagenesis cassettes lacked one of either Rep protein or the entire replicon (FIG. 6). Control gene targeting cassettes lacked one of either Rep protein or Cas9 (FIG. 7). Plants were grown in 1 liter pots in a monitored greenhouse in the Weizmann Institute of Science. Rehovot. The climate growth conditions were of 23° C., 70% humidity during the day and 19° C., 60% humidity during the night.

Isolation of DNA

DNA extraction was done by grinding two to three leaves in liquid nitrogen with an electric drill in a 1.5 ml Eppendorf tube. One volume of 700l pre-heated (to 65° C.) extraction buffer (0.8M NaCl, 0.15M Sorbitol, 0.12M Tris-HCl pH=7.5, 22 mM EDTA, 0.8% CTAB, 0.8% sodium lauryl sarcosinate) was added to each tube and incubated for 20 minutes in 65° C. One volume of 800 μl chloroform-octanol (24:1) was added to each tube and centrifuged at 13,000 rpm for 5 minutes. This step was repeated twice. One volume of 1.4 ml pre-cooled (4° C.) storage buffer (80% EtOH, 0.2M NaAcetate) was added to the supernatant in a new 2 ml Eppendorf tube. DNA precipitation was done by centrifugation at 13,000 rpm for 20 minutes in 4° C. The pellet was re-suspended in 50 μl of H₂O.

PCR Analysis

Two PCRs were performed using Taq Ready Mix (2×) (Hylabs) to confirm integration of the truncated CRTISO donor DNA to the tangerine locus. Each PCR was done with one primer from the genomic locus and one primer from the donor DNA 281 bp. To assess integration from the 5′ end, crtisoUpFw primer was used: 5′-CCTATGATCTAACATAATCTTGAAC (SEQ ID NO: 57) corresponding to the genomic locus and DelRv primer: 5′-GTGTGGCGAGGTATCAGAC (SEQ ID NO: 58) corresponding to the 281 bp complementation sequence. For integration from the 3′ end, DelFw primer: 5′-CACACCGCTCCATAGTCTG (SEQ ID NO: 59) corresponding to the 281 bp complementation sequence and crtiso4Rv primer: 5′-GTAACACATCTAAGTGTAGGGG (SEQ ID NO: 60) corresponding to the genomic locus were used (FIG. 8).

Two PCRs were done to check for heterozygosity of the T₀ plants that were positive for gene targeting. One PCR was done using two primers for each side of the deletion/complementation, crtiso2Fw primer: 5′-GCTTTGGGTGATAGCAAACC (SEQ ID NO: 61) and crtiso2Rv primer: 5′-GTGGACGGTTTACTGGAAAG (SEQ ID NO: 62). The second PCR was done using one primer upstream to the deletion/complementation, crtiso2Fw (SEQ ID NO: 61), and one primer corresponding to the genomic locus, crtiso4Rv (SEQ ID NO: 60). Long range PCR was done using TaKaRa LA Taq® in order to amplify unique fragments for GT using primers upstream and downstream to the donor and the 281 bp complementation (FIG. 9).

Southern Blot Analysis

Genomic DNA was extracted from Micro-Tom WT plant, from tangerine fast-neutron mutant, and from “TmicT2-donor-Rep-Ubi10” progeny of tangerine background plants with and without the gene targeting phenotype using Macherey-Nagel NucleoSpin® Plant II extraction kit. DNA was digested with NsiI and SspI-HF (New England Biolabs®) and fractionated on 0.8% agarose gels, transferred to nylon membranes (Hybond N+, Amersham), and hybridized overnight at 65° C. with 32P-labeled genomic 5′ and 3′ end probes as described in Results. The hybridization signal was obtained using a BIO-RAD phosphoimager PMI.

DNA Amplification and High-Throughput Sequencing

Genomic DNA extractions from gene targeting T₀ plants of all 8 constructs were used to produce amplicons for high-throughput (HT) deep sequencing. 18 cycles PCR on plant samples were done using KAPA HiFi HotStart ReadyMix (KAPA Biosystems-50 μl reactions according to the manufacturer's protocol). Primers were designed to amplify 274 bp of the CRTISO gene and 272 bp of the PSY1 gene, while the forward primer was 50-60 bp from the CRISPR target site. Primers had 6 degenerated bases (N) at the 5′ end (NNNNNN), and the forward primer had a 4 bases barcode to indicate the specific sample in each library (6 samples per library—3 of CRTISO, 3 of PSY1). The barcodes used for crtiso were GACT, TAGC and CGTA. and the barcodes used for PSY1 were ATCG, GTAC and CAGT (Table 5, barcodes are underlined). Samples of different constructs were distributed randomly in all libraries. Deep sequencing was done using Illumina sequencing HiSeq 2500 system, high output run, paired-end 125 bp, at the Nancy and Stephen Grand Israel National Center for Personalized Medicine (G-INCPM), the Weizmann Institute of Science.

TABLE 5 Primers used for amplicon amplification Name Sequence (5′ to 3′) SEQ ID NO: Crtiso HT Fw GACT NNNNNNGACTCTCTGCATCTATAAAAGACAGC 63 Crtiso HT Fw TAGC NNNNNNTAGCCTCTGCATCTATAAAAGACAGC 64 Crtiso HT Fw CGTA NNNNNNCGTACTCTGCATCTATAAAAGACAGC 65 Crtiso HT Rv NNNNNNTATTTAAGTGAAGAATGGTAAAGG 66 Psy1 HT Fw ATCG NNNNNNATCGGTATCGCCCCTGAATCAAAG 67 Psy1 HT Fw GTAC NNNNNNGTACGTATCGCCCCTGAATCAAAG 68 Psy1 HT Fw CGTA NNNNNNCAGTGTATCGCCCCTGAATCAAAG 69 Psy1 HT Rv NNNNNNAGTTCTGCAATTTTATTCCCAG 70

Mutations Detection by Sanger Sequencing

Various plant DNA samples of the gene targeting experiments were tested for CRISPR activity and NHEJ events by Sanger sequencing. Amplification of the target area was done using REDTaq® ReadyMix™ PCR Reaction Mix (Sigma-Aldrich), standard PCR protocol reaction on genomic DNA. After amplification detection by gel electrophoresis, positive reactions were put in an ExoI-Sap reaction to remove primers and nucleotides. Reaction total 9 μl: 5 μl PCR reaction, 1 μl Exonuclease I (New England BioLabs® Inc), 1 μl shrimp alkaline phosphatase (New England BioLabs® Inc), 2 μl DDW. The reaction was put for incubation in 37C for 30 minutes, followed by 10 minutes deactivation at 80′C. 10 mM primer were added to the reaction to a volume of 10 μl that was sent for Sanger sequencing at the DNA sequencing unit, Biological Services Department, the Weizmann Institute of Science with Applied Biosystems 3730 DNA Analyzer. PCRs for junction detection of the gene targeting plants were also sent to Sanger sequencing after the Exo-Sap reaction described above. Long range PCR fragments were extracted from gel using RBC HiYield™ Gel/PCR DNA Fragments Extraction Kit and were analyzed as described above. Due to low concentration, amplified 3′ junctions were subcloned to pGEM®-T Easy vector before sequencing.

Quantitative PCR

The relative number of replicons was detected by quantitative PCR (qPCR) analysis on various genomic DNA samples. The Fast SYBR® Green master mix (Applied Biosystems) was used for the DNA qPCR reactions, according to the manufacturer protocol in triplicate (3 technical replicates) 10 μl reactions. PCR amplification was tracked on StepOne™ system (Applied Biosystems). For the gene targeting system, a primer pair that can amplify the CRTISO donor was used (crtiso4Fw primer: 5′-TCTTTCACGCTGATGTGTGC (SEQ ID NO: 71) and crtiso3Rv primer: 3′-CTCTAGGACCCAACGACAGA (SEQ ID NO: 72)) and for the endogenous control the primer pair crtiso5Fw (5′-GGAGAACGAAGAGGGAAGAAC (SEQ ID NO: 73)) and crtiso4Rv were used (that are not on the CRTISO donor). The amplification efficiencies of all primer pairs were approximately equal (Calibration of primers: crtiso4F-3R slope −3.45, R² 0.99: crtiso5F-4R slope −3.47, R² 0.99).

Example 5 Gene Targeting

Objective:

To improve targeted mutation rates by using stable and high expression levels of the CAS9 gene. The experiments and results presented here expand those presented in Example 3. Some of the results of Example 3 are included here.

Methods:

Two different promoters were tested for constitutive expression of the Cas9 endonuclease. Ubiquitin 4-2 from Petroselinum crispum (“Ubi4”, as used in Steinert et al. Plant J. (2015) 84, 1295-1305) and Ubiquitin 10 from Solanum lycopersicum (“Ubi10”). Two genes were targeted, the carotenoid isomerase (CRTISO) gene (Solyc10g081650), and phytoene synthase 1 (PSY1) gene (Solyc03g031860) from the carotenoid biosynthesis pathway, due to their distinct fruit phenotype (Enfissi et al. Plant J. (2016), 89, 774-788). The crtiso mutant has orange fruits, yellowish young leaves and pale petals, while the psy1 mutant has a yellow fruit but other organs are wild type (Isaacson et al., Plant Cell (2002), 14, 333-42). Both are recessive mutations, in which homozygous or bi-allelic mutant result in a non-WT phenotype.

In addition, it was tested whether the amplification of Cas9 and gRNA through geminiviral replicon rolling circle replication (RCR), enhanced the DSB rate as seen through the analysis of non-homologous end-joining (NHEJ) DSB repair events. All constructs used in these experiments are shown in FIG. 6.

Early stage results are results collected after moving the plant from tissue culture to soil and before the flowering of the plant. Late stage results are those results encompassing reproductive growth. DNA sequence analysis was performed using Illumina sequencing HiSeq 2500 system, high output run, paired-end 125 bp at the G-INCPM.

Results:

Mutated phenotypes were easy to detect already in the T₀ transformed plant. Many plants had all their fruits fully mutated, i.e. orange fruit color phenotype of crtiso or yellow fruits corresponding to the psy1 mutation (FIG. 10A), indicating early mutagenesis events. Some plants had a “chimeric” phenotype, i.e. a mixture of WT (red color) fruits, yellow or orange (tangerine) fruits (depending on the target) and sectored fruits, indicating late somatic mutations. In the case of early mutation events, plants with the CRISPR/Cas9 system targeting the CRTISO gene had late greening of the young leaves phenotype as early as 2 days after moving to the greenhouse (FIG. 10B). A further phenotype of the crtiso mutation was the pale orange petals (FIG. 10C).

Only plants that were positive for transfer DNA (T-DNA) integration and produced fruits were considered in the phenotypic analysis. The phenotype was scored as red, orange or yellow if all the fruits of the T₀ plant were red, orange or yellow respectively. T₀ plants were considered as chimeras if they bear a mixture of WT (red) or mutant (orange or yellow) or sectored fruits. Remarkably, for the CRTISO target most constructs showed similar and very high frequencies of mutagenesis having close to 100% mutations in both alleles. For the PSY1 target, the frequency of mutagenesis ranged between 70-50%. Despite the presence of the geminiviral replicon, no mutagenesis enhancing effect was observed. Plant phenotypes for each transformant are summarized in Table 3 (see Example 3)

Conclusion:

Constructs targeted against crtiso showed very high frequencies of mutagenesis. These frequencies were similar for all tested crtiso constructs. The presence of a geminiviral replicon did not enhance the efficiency of constructs targeted against psy1.

Example 6 Detection of Mutants by Next Generation Sequence

Objective:

To determine the frequencies and sequences of the mutation footprints for the CRTISO and PSY1 targets.

Methods:

Tissue from true leaves of T₀ plants transformed with the eight different constructs shown in FIG. 6 was collected and their DNA was extracted. The region around the CRTISO or PSY1 target was PCR-amplified and sent to deep sequencing using Illumina Hi-Seq for detection of mutations. Overall, results were obtained from 156 plants; 76 plants with gRNA targeting the CRTISO gene and 80 plants with gRNA targeting the PSY1 gene. For negative control, amplicons were obtained from Micro-Tom plants expressing only the Cas9 under Ubi10 promoter (no gRNA). Plants with more than >97% of the Illumina reads different from WT were considered as fully mutated, since minor amount of WT reads might correspond to contaminations or very minor chimerism from the calli. This assumption is supported by the phenotypic data showing plants containing only mutant fruits.

Results:

The frequencies and sequence of the various mutation footprints are shown in FIGS. 11A and 11B for the CRTISO and PSY1 targets respectively. Mutation frequencies are higher in plants targeting the CRTISO gene, when most of the plants had only up to 4 different repair footprints per plant. The tomato plants are diploid, therefore a multiplicity of footprints probably suggests that plants are chimeric with different types of mutations being formed during development.

Conversely, the presence of one or two types of footprints, in the absence of the WT sequence, suggests that early events occurred and were clonally inherited in all cells. Plants with bi-allelic mutations were often observed, and plants that were homozygous to a certain type of footprint, for example (−T) and (−AGCAT) mutation events (FIG. 11A, plants Ubi4 #9 and Ubi10 #2, respectively). Fewer mutation events were obtained with the psy1 gRNA, while a (−G) deletion was commonly observed, with one plant that appears to be homozygous for this repair footprint (FIG. 11B, plant rep Ubi4 38). Overall 64 plants out of 76 were fully mutated in the CRTISO gene (84%) compared to 15 plants out of 80 that were fully mutated in the psy1 gene (19%). Across all analyzed plants, 90.4% and 56.4% of the WT alleles were mutated in CRTISO and PSY1 loci respectively. In order to test transmission to the next generation T₁ progeny of T₀ plants that had lost the WT allele were grown. All T₁ progeny had a mutant phenotype. Sequencing of crtiso in two T₁ progeny of T₀ plant crtiso-Ubi10 #18 which had three mutated alleles (FIG. 11A), gave rise to one plant homozygous for −1 T deletion, and another plant heterozygote for −1 T deletion and a −9 (ACCAGCATT) deletion. These results show efficient mutagenesis rates and transmission to the next generation.

Conclusions:

Gene targeting using the CRISPR-Cas9 system was extended to a new locus (CRTISO). Mutation frequencies were very high at the CRTISO locus, altering 90.4% of the WT alleles in T₀ plants. Sequence analysis from leaf tissues of T₀ plants showed little chimerism, with most plants containing only one or two mutant alleles, suggesting very early CRISPR-Cas9 mutagenesis in the somatic embryo. Mutagenesis frequency was lower for the PSY1 locus. Considering that all components used for PSY1 mutagenesis were similar to those used for CRTISO, the gRNA design or the epigenetic context must be the reason for low mutagenesis at PSY1 locus.

Example 7 Repairing of the Crtiso Allele—Gene Replacement

Objective:

To replace a defective endogenous gene with a wildtype sequence, with no selection for homologous recombination during the tissue culture stage.

Methods:

A tangerine Micro-Tom mutant with a 281 bp deletion in the crtiso gene, generated using fast-neutron mutagenesis (Meissner et al. Plant J. (1997), 12, 1465-1472) and described by Isaacson et al., Plant Cell (2002), 14, 333-42, was chosen as a target in order to perform a knock-in experiment whereby the deletion in the crtiso gene is replaced by the WT sequence. The system combines induction of a DSB by a CRISPR/Cas9 system in the site of the tangerine deletion, together with delivery of a geminiviral replicon that carries a 3796 bp donor repair template. Phenotypically, the replacement of the defective tangerine allele by the WT donor sequence was expected to result in a red WT fruit phenotype. A repair in one of the alleles is sufficient for developing red fruits since the tangerine mutation is recessive. FIG. 12 shows the gene targeting strategy used in these experiments. Plants were transformed with a single construct, from those depicted in FIG. 7, containing Cas9 under the expression of the SlUbiquitin10 (Ubi10) promoter, a gRNA (U6-26:tangerine t1 or t2), the Rep protein, a CRTISO gene that contains the WT coding region that is deleted in the tangerine mutant and serves as the donor repair template. The donor repair template is amplified using the geminiviral replicon. A DSB (lightening symbol) is induced in the tangerine mutant deletion allele using the CRISPR/Cas9 system. Recombination in the regions of homology between the donor and the broken target (1778 bp and 1737 bp 5′ and 3′ regions respectively) results in the replacement of the deletion allele by the WT sequence (red box), leading to a red instead of tangerine fruit color phenotype.

The transfer DNA (T-DNA) constructs used in the gene targeting experiment (“TmicT1-donor-Rep-Ubi10” and “TmicT2-donor-Rep-Ubi10”, FIG. 7) harbors both the CRISPR/Cas9 system, with Cas9 expressed under the Ubi10 promoter, the replicon machinery, and two different gRNAs. “T1” and “T2”. CRISPR gRNAs were designed to match target sequences only in the tangerine allele. These target sequences become destroyed, or inactivated, upon insertion of the repair donor sequence. T1 targets a sequence adjacent to the CRIPSR mandatory PAM sequence. Therefore, T1 target sequence is destroyed upon repair, since the inserted donor sequence separates PAM from the target sequence. T2 targets a sequence flanking the tangerine deletion site. Therefore, upon repair, T2 target sequence is destroyed since the donor sequence is inserted within it (FIG. 13).

TmicT1-donor-Rep-Ubi10 and TmicT2-donor-Rep-Ubi10 constructs enabled replicon amplification together with DSB induction already in the T₀ plant, giving rise to 32 and 36 transformed T₀ plants with ripe fruits, respectively. Plants were also transfected with control constructs. Table 6 shows the construct used in the experiments (FIG. 7) and the number of plants with ripe fruits for each of them.

TABLE 6 Phenotypic analysis of gene targeting of the CRTISO and PSY1 loci in Micro-Tom Wild Type plants Construct name # of plants TmicT1-donor-Rep-Ubi10 32 TmicT2-donor-Rep-Ubi10 36 TmicT1-donor-Rep 11 TmicT2-donor-Rep 12 TmicT1-donor-Ubi10 13 TmicT2-donor-Ubi10 12 TmicT1-donor 10 TmicT2-donor 17 Rep-Ubi10 7 Rep 2 Ubi10 5

While tangerine mutants have late greening of the young leaves phenotype, it is not a reliable marker being very much affected by light growth conditions. Therefore, it was not taken into account when selecting kanamycin-resistant calli for regeneration in the treatment or in the controls.

Results:

The presence of replicons in transfected cells was measured by DNA quantitative PCR of plants transfected either with the full cassette constructs including Rep, or with their CRISPR-only controls. Replicon amplification was measured by using primers annealing to both the tangerine locus and the CRTISO donor, the tangerine mutant served as the baseline copy number. Four groups were tested: “TmicT1-donor-Rep-Ubi10” and “TmicT2-donor-Rep-Ubi10” contained all the components for the GT experiment, compared to plants without the replicon “TmicT1-donor-Ubi10” and “TmicT2-donor-Ubi10”, which served as negative controls. Each treatment had 3 biological replicates. Results showed up to 90-fold increase in replicon copy number in comparison to the endogenous control (FIG. 14).

Nine (9) out of the 36 tested T₀ plants that were transformed with “TmicT2-donor-Rep-Ubi10” were found to have only red fruits (25%). T₁ progeny of eight of these nine plants were grown, showing Mendelian segregation for the red/orange phenotype, indicating that T₀ plants were heterozygous and showing transmission to the next generation. The ninth plant was highly sterile and a single seed was obtained giving rise to a single T₁ plant with orange fruits. 1 out of the 12 tested T₀ plant that were transformed with control “Tmic-T2-donor-Ubi10” construct devoid of replicon activity, gave rise to a chimeric plant with red and orange fruits. The T₁ progeny of orange fruits gave rise to orange fruits and the T₁ progeny of the red fruits segregated for the red/orange phenotype, indicating that the red fruits from T₀ plants were heterozygous. All other experiments and control constructs had only orange fruits.

In order to confirm that the red fruits were the product of “TmicT2-donor-Rep-Ubi10” gene targeting, validation was done by PCR amplification of DNA extracted from leaf tissue of the 9 T₀ plants having only red fruits. Primers were designed to amplify the gene target event, but not the tangerine locus. To identify integration at the 5′ end of the CRTISO donor, PCR was done using one forward primer from the 5′ of the crtiso locus (crtisoUpFw) that does not exist on the donor and a second reverse primer from the WT 281 bp complementation (delRv) that does not exist on tangerine (FIG. 15A). PCR products of 8 GT plants were checked next to Micro-Tom WT as positive control, tangerine mutant and no template (blank) as negative controls. PCR products were separated by electrophoresis in an EtBr-stained agarose gel, showing that the size of the amplified fragments corresponded to the expected 2122 bp length of the repaired sequence 8 GT plants were checked next to Micro-Tom WT as positive control, and tangerine mutant and no template (blank) as negative controls (FIG. 15B). To identify integration at the 3′ end, primers delFw (not present on tangerine) and crtiso4Rv (from the 3′ of the crtiso locus) were used (FIG. 15A), yielding the expected PCR fragment size of 1971 bp for a GT allele (FIG. 15C). To check if the red-fruit plants are homozygous or heterozygous, the tangerine allele or the WT allele (originated from repaired allele or donor sequence), were PCR amplified with 2 pairs of primers flanking the tangerine deletion. The first pair of primers, crtiso2Fw and crtiso2Rv, amplified either the tangerine allele (668 bp), the WT repaired allele (949 bp), or both (FIG. 9). The second pair of primers, crtiso2Fw and crtiso4Rv, amplified either the tangerine allele (2235 bp), the WT repaired allele (2516 bp), or both (FIG. 9). PCR products were loaded into an EtBr agarose gel and separated by electrophoresis. Electrophoresis revealed 668 bp and 949 bp PCR products amplified with the first pair of primers (FIG. 16A), and 2235 bp and 2516 bp PCR products amplified with the second pair of primers (FIG. 16B), showing T₀ plants were heterozygous.

The progeny of 4 T₀ plants with gene targeting phenotype was analyzed by Southern blot hybridization. Southern blot was done on genomic DNA digested by NsiI and SspI-HF, to detect 5′ and 3′ integration respectively. FIG. 17A shows the location of the NsiI and SspI-HF restriction sites and of the probes (red rectangles) used in the Southern blot analysis. Southern blot of Micro-Tom WT DNA (lane 1) showed only the expected WT NsiI 5083 bp/SspI 2964 bp fragment, while tangerine mutant DNA (lane 2) and tangerine mutant that had the T-DNA but did not undergo GT (lane 3) showed the expected NsiI 6311 bp/SspI 3964 bp fragments (FIG. 17B). GT progeny plants (GT1, GT4, GT5, corresponding to lanes 4, 6 and 7 respectively), showed heterozygosity at their CRTISO locus with both tangerine and WT repaired alleles fragments. GT2 progeny plant was homozygous for the WT repaired allele.

PCR fragments were amplified from upstream and downstream of the WT 281 bp complementation region to the CRTISO locus using TaKaRa LA Taq® (FIGS. 18A and 18B). Sanger sequencing of the amplified fragments were found to have the expected sequence for homologous recombination between the donor sequence and the 5′ and 3′ junctions of the locus (FIG. 8).

In order to understand absence of repair in transfected plants, Sanger sequencing was performed in T₀ plants transfected with the TmicT1-donor-Rep-Ubi10 and TmicT2-donor-Rep-Ubi10 construct still having the tangerine phenotype after transfection. FIG. 19 shows the sequences obtained from a not-transformed tangerine mutant (upper panel), plants #3 and #1 transformed with TmicT1-donor-Rep-Ubi10 (two middle panels), and plant #2 transformed with TmicT2-donor-Rep-Ubi10. Non-homologous end joining (NHEJ) was detected in target region T1 and target region T2. Importantly. NHEJ repair at this site prevent a second DSB and repair by homologous recombination.

Conclusion:

A very high rate of gene targeting was achieved in the absence of selection for the targeting event. In addition, gene targeting, namely the replacement of the deleted tangerine allele by the WT sequence, occurred very early in development as inferred from the finding that all the fruits of the GT T₀ plants (9 out of 36) were red (WT) and that this phenotype was germinally transmitted. This is consistent with the early timing of CRISPR-Cas9-induced DSB induction. Considering that Cas9 cannot cleave its DNA target again after error-prone DSB break occurs, it is necessary to have a precise coordination of the DSB induction together with the donor amplification to increase the chances of template invasion during the homologous-recombination-mediated repair. Moreover, all steps in the process must be optimal for gene targeting (rather than non-homologous end joining) to take place, such as strong Cas9 expression (here under the tomato Ubi10 promoter), an effective gRNA and coordinated amplification of the geminiviral replicon, in this case all components were cloned in a single construct and were co-expressed.

These experiments are an unexpected advance over earlier reports, showing the feasibility of selection-free and efficient gene targeting. Only selection by Kanamycin resistance at the stage of transformation was used, and plants were grown with no knowledge on their phenotype until flowering (white petals in tangerine versus yellow in WT) and fruit ripening (orange versus red).

Example 8 Repairment of the Crtiso Allele is not Due to Random Contamination

Objective:

To validate that plants are repaired by CRISPR/Cas9 and not by seed or pollen contamination.

Methods:

The WT Micro-Tom fruit has a glossy and red fruit surface. By contrast, the tangerine mutant has a non-glossy surface and orange color, as a consequence of the several mutations in its background (Meissner et al. (1997) ibid) (FIG. 20). Surfaces of tomatoes were observed in tangerine mutant plants targeted or not with the CRISPR/Cas9 construct.

Results:

Glossy red surfaces were observed in WT plants. The glossiness was maintained in CRTISO mutated plants, showing that CRTISO mutations do not affect glossiness. Non-glossy yellow surfaces were observed in tangerine mutants. While tangerine mutants having the CRISPR/Cas9 construct showed a repaired red surface, the non-glossy phenotype was maintained (FIG. 20).

Conclusion:

Gene targeting occurred in the tangerine background by the CRISPR/Cas9 construct and could not be due to a contamination, as plants with red non-glossy phenotype did not exist in the mutant collection.

While certain features of methods of gene targeting and gene replacement have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes. 

What is claimed is:
 1. A method for gene targeting in a plant cell, the method comprising: (a) introducing into said plant cell a first nucleic acid comprising a viral replicon comprising a donor nucleic acid sequence, said donor sequence targeted to a plant endogenous DNA sequence; and (b) introducing into said same plant cell a second nucleic acid comprising a nuclease system, wherein said nuclease system is targeted to said plant endogenous DNA sequence, and wherein at least one component of said nuclease system is expressed under a UBQ10 regulatory sequence; wherein homologous recombination occurs between the donor sequence and said plant endogenous DNA sequence.
 2. The method of claim 1, wherein said viral replicon is selected from a group comprising: a geminiviral replicon, a bean yellow dwarf virus (BeYDV) replicon, a cabbage leaf curl virus (CalCuV) replicon, a tomato leaf curl virus (ToLCV) replicon, a wheat dwarf virus (WDV) replicon, or any combination thereof.
 3. The method of claim 1, wherein said donor sequence comprises a gene, a mutated gene, a part of a gene, a regulatory sequence, a mutated regulatory sequence, a sequence upstream of a gene, a sequence downstream of a gene, an exon sequence, an intron sequence, or any combination thereof.
 4. The method of claim 1, wherein said nuclease system is selected from a group comprising: a nickase, a zinc finger nuclease (ZFN) system, a transcription activator-like effector nuclease (TALEN) system, a meganuclease, or a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) nuclease.
 5. The method of claim 4, wherein said CRISPR/Cas nuclease system comprises a CRISPR-associated endonuclease (Cas) and a gRNA molecule, wherein said gRNA molecule binds within said plant endogenous DNA sequence.
 6. The method of claim 5, wherein said Cas enzyme is selected from the group comprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, C2c1, CasX, NgAgo, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csm2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4, or homologs thereof, or modified versions thereof.
 7. The method of claim 5, wherein said Cas is operably linked to said UBQ10 regulatory sequence.
 8. The method of claim 1, wherein a single expression vector comprises said first nucleic acid and said second nucleic acid.
 9. The method of claim 1, wherein said UBQ10 regulatory sequence is selected from a group comprising: Solanum lycopersicum, Solanum tuberosum, or Arabidopsis thaliana UBQ10 regulatory sequence.
 10. The method of claim 9, wherein said Solanum lycopersicum UBQ10 regulatory sequence comprises Solanum lycopersicum UBQ10 promoter and terminator regions.
 11. The method of claim 1, wherein said gene targeting comprises gene editing, gene replacement, or a combination of both.
 12. A recombinant nucleic acid molecule comprising a first nucleotide sequence encoding a nuclease system, wherein said nuclease system is targeted to a plant endogenous DNA sequence, and wherein at least one component of said nuclease system is operably linked to a UBQ10 regulatory sequence.
 13. The recombinant nucleic acid molecule of claim 12 further comprising a second nucleotide sequence encoding a viral replicon comprising a donor nucleic acid sequence targeted to said plant endogenous DNA sequence.
 14. The recombinant nucleic acid of claim 13, wherein said viral replicon is selected from a group comprising: a geminiviral replicon, a bean yellow dwarf virus (BeYDV) replicon, a cabbage leaf curl virus (CalCuV) replicon, a tomato leaf curl virus (ToLCV) replicon, a wheat dwarf virus (WDV) replicon, or any combination thereof.
 15. The recombinant nucleic acid of claim 14, wherein said donor sequence comprises a gene, a mutated gene, a part of a gene, a regulatory sequence, a mutated regulatory sequence, a sequence upstream of a gene, a sequence downstream of a gene, an exon sequence, an intron sequence, or any combination thereof.
 16. The recombinant nucleic acid of claim 12, wherein said nuclease system is selected from a group comprising: a nickase, a zinc finger nuclease (ZFN) system, a transcription activator-like effector nuclease (TALEN) system, a meganuclease, or a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) nuclease.
 17. The recombinant nucleic acid of claim 16, wherein said CRISPR/Cas nuclease system comprises a CRISPR-associated endonuclease (Cas) and a gRNA molecule, wherein said gRNA molecule binds within said plant endogenous DNA sequence.
 18. The recombinant nucleic acid of claim 17, wherein said Cas enzyme is selected from the group comprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, C2c1, CasX, NgAgo, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4, or homologs thereof, or modified versions thereof.
 19. The recombinant nucleic acid of claim 18, wherein said Cas nuclease is operably linked to said UBQ10 regulatory sequence.
 20. The recombinant nucleic acid of claim 19, wherein said UBQ10 regulatory sequence is selected from a group comprising: Solanum lycopersicum, Solanum tuberosum, or Arabidopsis thaliana UBQ10 regulatory sequence.
 21. The recombinant nucleic acid of claim 20, wherein said Solanum lycopersicum UBQ10 regulatory sequence comprises Solanum lycopersicum UBQ10 promoter and terminator regions.
 22. A method for producing a transgenic plant seed, the method comprising: (a) introducing into at least one cell of a plant a first nucleic acid comprising a viral replicon comprising a donor nucleic acid sequence, said donor sequence targeted to a plant endogenous DNA sequence; and (b) introducing into the cell of (a) a second nucleic acid comprising a nuclease system, wherein said nuclease system is targeted to said plant endogenous DNA sequence, and wherein at least one component of said nuclease system is expressed under a UBQ10 regulatory sequence; (c) generating a transgenic plant from said at least one cell; and (d) growing said transgenic plant to obtain a seed; wherein homologous recombination occurs between the donor sequence and said plant endogenous DNA sequence: thereby producing a transgenic seed of a plant, wherein any plant produced from said seed comprises said donor nucleic acid sequence.
 23. The method of claim 22, wherein said viral replicon is selected from a group comprising: a geminiviral replicon, a bean yellow dwarf virus (BeYDV) replicon, a cabbage leaf curl virus (CalCuV) replicon, a tomato leaf curl virus (ToLCV) replicon, a wheat dwarf virus (WDV) replicon, or any combination thereof.
 24. The method of claim 22, wherein said donor sequence comprises a gene, a mutated gene, a part of a gene, a regulatory sequence, a mutated regulatory sequence, a sequence upstream of a gene, a sequence downstream of a gene, an exon sequence, an intron sequence, or any combination thereof.
 25. The method of claim 22, wherein said nuclease system is selected from a group comprising: a nickase, a zinc finger nuclease (ZFN) system, a transcription activator-like effector nuclease (TALEN) system, a meganuclease, or a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) nuclease.
 26. The method of claim 25, wherein said CRISPR/Cas nuclease system comprises a CRISPR-associated endonuclease (Cas) and a gRNA molecule, wherein said gRNA molecule binds within said plant endogenous DNA sequence.
 27. The method of claim 26, wherein said Cas enzyme is selected from the group comprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, C2c1, CasX, NgAgo, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4, or homologs thereof, or modified versions thereof.
 28. The method of claim 26, wherein said Cas is operably linked to said UBQ10 regulatory sequence.
 29. The method of claim 22, wherein said first nucleic acid and said second nucleic acid are located on a single expression vector.
 30. The method of claim 22, wherein said UBQ10 regulatory sequence is selected from a group comprising: Solanum lycopersicum, Solanum tuberosum, or Arabidopsis thaliana Ubi10 regulatory sequence.
 31. The method of claim 30, wherein said Solanum lycopersicum UBQ10 regulatory sequence comprises Solanum lycopersicum UBQ10 promoter and terminator regions. 